Quantitative proteomic analysis of ahpC/F and katE and katG knockout Escherichia coli-a useful model to study endogenous oxidative stress.

Alkyl hydroperoxide reductase (AhP), catalase G (KatG), and catalase E (KatE) are the main enzymes to scavenge the excessive hydrogen peroxide in E. coli. It was found the concentration of endogenous HO was submicromolar in a mutant strain E. coli MG1655/ΔAhpΔKatEΔKatG, which was enough to cause damage to DNA and proteins as well as concomitant cell growth and metabolism. However, few studies explored how submicromolar intracellular hydrogen peroxide alters protein function and regulates the signaling pathways at the proteome level. In order to study the effect of endogenous oxidative stress caused by submicromolar hydrogen peroxide, this study first constructed a mutant strain E. coli MG1655/ΔAhpΔKatEΔKatG. Then, label-free quantitative proteomic analysis was used to quantify the differentially expressed proteins between the wild-type strain and the mutant strain. A total of 265 proteins were observed as differentially expressed proteins including 108 upregulated proteins and 157 downregulated proteins. Among them, three differentially expressed proteins were also validated by parallel reaction monitoring (PRM) methodology. The 265 differentially expressed proteins are not only involved with many metabolism pathways including the TCA cycle, the pentose phosphate pathway, and the glyoxylic acid cycle, but also activated the DNA repair and cellular antioxidant signaling pathway. These findings not only demonstrated that ahp, katE, and katG played the critical role in aerobic growth but also delineated proteins network and pathway regulated by submicromolar intracellular hydrogen peroxide, which allowed a deeper understanding of oxidative signaling in E. coli. The findings of this study also demonstrate that the mutant E. coli may serve as a cell model to investigate the effect of endogenous oxidative stress and downstream signaling pathways. KEY POINTS: • The mutant strain E. coli MG1655/ΔAhpΔKatEΔKatG was constructed to study the effect of endogenous oxidative stress in E. coli. • A total of 265 differentially expressed proteins were quantified and enriched in metabolic pathways and antioxidant systems by using label-free proteomics analysis. • The findings of this study demonstrate that the mutant E. coli may serve as an effective tool to investigate the endogenous oxidative stress.