Translational systems can respond promptly to sudden environmental changes to provide rapid adaptations to environmental stress. Unlike the well-studied translational responses to oxidative stress in eukaryotic systems, little is known regarding how prokaryotes respond rapidly to oxidative stress in terms of translation. In this study, we measured protein synthesis from the entire Escherichia coli proteome and found that protein synthesis was severely slowed down under oxidative stress. With unchanged translation initiation, this slowdown was caused by decreased translation elongation speed. We further confirmed by tRNA sequencing and qRT-PCR that this deceleration was caused by a global, enzymatic downregulation of almost all tRNA species shortly after exposure to oxidative agents. Elevation in tRNA levels accelerated translation and protected E. coli against oxidative stress caused by hydrogen peroxide and the antibiotic ciprofloxacin. Our results showed that the global regulation of tRNAs mediates the rapid adjustment of the E. coli translation system for prompt adaptation to oxidative stress.

Streptococcus pneumoniae (S.pneumoniae) is a major human pathogen causing morbidity and mortality worldwide. Efficiently acquiring iron from the environment is critical for S. pneumoniae to sustain growth and cause infection. There are only three known iron-uptake systems in Streptococcal species responsible for iron acquisition from the host, including ABC transporters PiaABC, PiuABC, and PitABC. Besides, no other iron-transporting system has been suggested. In this work, we employed our newly established translating mRNA analysis integrated with proteomics to evaluate the possible existence of novel iron transporters in the bacterium. We simultaneously deleted the iron-binding protein genes of the three iron-uptake systems to construct a piaA/piuA/pitA triple mutant (Tri-Mut) of S. pneumoniae D39, in which genes and proteins related to iron transport should be regulated in response to the deletion. With ribosome associated mRNA sequencing-based translatomics focusing on translating mRNA and iTRAQ quantitative proteomics based on the covalent labeling of peptides with tags of varying mass, we indeed observed a large number of genes and proteins representing various coordinated biological pathways with significantly altered expression levels in the Tri-Mut mutant. Highlighted in this observation is the identification of several new potential iron-uptake ABC transporters participating in iron metabolism of Streptococcus. In particular, putative protein SPD_1609 in operon 804 was verified to be a novel iron-binding protein with similar function to PitA in S. pneumoniae. These data derived from the integrative translatomics and proteomics analyses provided rich information and insightful clues for further investigations on iron-transporting mechanism in bacteria and the interplay between Streptococcal iron availability and the biological metabolic pathways.

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Sodium new houttuyfonate (SNH), an addition product of active ingredient houttuynin from the plant Houttuynia cordata Thunb., inhibits a variety of bacteria, yet the mechanism by which it induces cell death has not been fully understood. In the present study, we utilized iTRAQ-based quantitative proteomics to analyze the protein alterations in Streptococcus pneumoniae in response to SNH treatment. Numerous proteins related to the production of reactive oxygen species (ROS) were found to be up-regulated by SNH, suggesting that ROS pathways may be involved as analyzed via bioinformatics. As reported recently, cellular reactions stimulated by ROS including superoxide anion (O2(•-)), hydrogen peroxide (H2O2), and hydroxyl radicals (OH(•)) have been implicated as mechanisms whereby bactericidal antibiotics kill bacteria. We then validated that SNH killed S. pneumoniae in a dose-dependent manner accompanied by the increasing level of H2O2. On the other hand, the addition of catalase, which can neutralize H2O2 in cells, showed a significant recovery in bacterial survival. These results indicate that SNH indeed induced H2O2 formation to contribute to the cell lethality, providing new insights into the bactericidal mechanism of SNH and expanding our understanding of the common mechanism of killing induced by bactericidal agents.

Trans-Cinnamaldehyde (TC) is a widely used food additive, known for its sterilization, disinfection, and antiseptic properties. However, its antibacterial mechanism is not completely understood. In this study, quantitative proteomics was performed to investigate differentially expressed proteins (DEPs) in Escherichia coli in response to TC treatment. Bioinformatics analysis suggested aldehyde toxicity, acid stress, oxidative stress, interference of carbohydrate metabolism, energy metabolism, and protein translation as the bactericidal mechanism. E. coli BW25113ΔyqhD, ΔgldA, ΔbetB, ΔtktB, ΔgadA, ΔgadB, ΔgadC, and Δrmf were used to investigate the functions of DEPs through biochemical methods. The present study revealed that TC exerts its antibacterial effects by inducing the toxicity of its aldehyde group producing acid stress. These findings will contribute to the application of TC in the antibacterial field.