Transcriptomic Analysis of E. coli after Exposure to a Sublethal Concentration of Hydrogen Peroxide Revealed a Coordinated Up-Regulation of the Cysteine Biosynthesis Pathway

Abstract

Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor OxyR leading to up-regulation of antioxidant systems, including the catalase-encoding gene (katG), that rapidly degrade extracellular H2O2 and promote bacterial survival. We documented a strong and transient up-regulation of genes involved in sulfur metabolism and cysteine biosynthesis, which are under the control of the transcription factor CysB. This strong specific transcriptional response to H2O2 exposure had no apparent impact on bacterial survival, but possibly replenishes the stores of oxidized cysteine and glutathione. In summary, our results demonstrate that different stress response mechanisms are activated by H2O2 exposure and highlight the cysteine synthesis as an antioxidant response in E. coli.

Transposon-Directed Insertion-Site Sequencing Reveals Glycolysis Gene gpmA as Part of the H2O2 Defense Mechanisms in Escherichia coli

“…In a previous study, we performed a RNA-seq analysis of E. coli BW25113 after a 10 min exposure to a sublethal concentration (2.5 mM) of H 2 O 2 [ 10 ]. Among the ten genes identified by TraDIS, only two genes, dps and gpmA, were significantly dysregulated by H 2 O 2 ( Figure 4 A).…”

“…To assess the susceptibility to H 2 O 2 and other oxidants, we used disk diffusion assay as previously described [ 10 ]. Briefly, an overnight culture of bacteria was diluted in LB to McFarland 0.5 using a Densimat (bioMérieux, Marcy-l’Étoile, France) and LB agar plates were inoculated using a sterile cotton swab.…”

“…Quantitative PCR (qRT-PCR) was performed on RNA samples as previously described [ 10 ]. Briefly, the cDNA was produced by reverse-transcribing 500 ng of total RNA using a mix of random hexamers and oligo d(T) primers and Primescript reverse transcriptase enzyme (Takara Bio, Saint Germain-en-Laye, France).…”

“…( A ) Differential expression of the 10 genes identified by TraDIS 10 min after exposition to 2.5 mM H 2 O 2 compared to no treatment (mean +/− SD, N = 4). Data originated from previously performed RNA-seq (deposited on ENA with the accession number: PRJEB51098) [ 10 ]. ( B , C ) Levels of expression of katG and gpmA, respectively, in the strain MG1655 under increasing concentration of H 2 O 2 , assessed by qRT-PCR, ** corresponds to p < 0.01.…”

“…The effect of H 2 O 2 on bacteria has been partially studied, but a complete picture of how H 2 O 2 affects bacteria and the bacterial response has not been elucidated for any bacterial species. Previous studies on H 2 O 2 tolerance, using DNA microarrays and RNA-seq, identified genes regulated under H 2 O 2 exposure [ 8 , 9 , 10 ] in E. coli . These studies permitted a better understanding of the regulation of numerous genes and pathways affected by H 2 O 2 exposure.…”