Characterizing Common Factors Affecting Replication Initiation During H2O2 Exposure and Genetic Mutation-Induced Oxidative Stress in Escherichia coli

Abstract

Oxidative stress is prevalent in organisms, and excessive oxidative damage can trigger cell death. Bacteria have evolved multiple pathways to cope with adverse stress, including the regulation of the cell cycle. Previous studies show that non-lethal exposure to H₂O₂ and mutations in antioxidant enzymes suppress replication initiation in Escherichia coli. The existence of common regulatory factors governing replication initiation across diverse causes-induced oxidative stress remains unclear. In this study, we utilized flow cytometry to determine the replication pattern of E. coli, and found that oxidative stress also participated in the inhibition of replication initiation by a defective iron regulation (fur-bfr-dps deletion). Adding a certain level of ATP promoted replication initiation in various antioxidant enzyme-deficient mutants and the ΔfurΔbfrΔdps mutant, suggesting that low ATP levels could be a common factor in the inhibition of replication initiation by different causes-induced oxidative stress. More potential common factors were screened using proteomics, followed by genetic validation with H₂O₂ stress. We found that oxidative stress might mediate the inhibition of replication initiation by interfering with the metabolism of glycine, glutamate, ornithine, and aspartate. Blocking CcmA-dependent cytochrome c biosynthesis, deleting the efflux pump proteins MdtABCD and TolC, or the arabinose transporter AraFHG eliminated the replication initiation inhibition by H₂O₂. In conclusion, this study uncovers a common multifactorial pathway of different causes-induced oxidative stress inhibiting replication initiation. Dormant and persistent bacteria exhibit an arrested or slow cell cycle, and non-lethal oxidative stress promotes their formation. Our findings contribute to exploring strategies to limit dormant and persistent bacterial formation by maintaining faster DNA replication initiation (cell cycle progression).

Keywords: ATP levels; DNA replication initiation; efflux pumps; metabolism; oxidative stress.

Figure 1

The effect of iron metabolism on replication initiation is associated with oxidative stress. (A,C) DNA replication patterns of E. coli. Exponentially growing E. coli cells were cultured to OD₄₅₀ = 0.15–0.2 in ABTG-CAA medium at 37 °C and treated with rifampicin and cephalexin for 3–5 doubling times. E. coli cells were fixed with 70% ethanol and then incubated with Hoechst 33,258 fluorescent dye; 10,000 cells were analyzed for DNA replication patterns using flow cytometry. The number of chromosome equivalents contained per cell is indicated on the X–axis, and the number of cells is shown on the Y–axis. In panel (A), doubling times are labeled in boxes and indicated by D.T. In panel (C), the indicated concentrations of ATP were added at around OD₄₅₀ = 0.04 and co-incubated with the culture for about two doubling times to grow to OD₄₅₀ = 0.15–0.2. (B,D) The average number of replication origins contained per bacterial cell in the respective category in panel (A,C). The average number of replication origins per cell (A.O.) represents the sum of the products of the number of chromosomes and the percentage of related cells. Data are the average of three independent biological replicates (marked above the corresponding column plot and showing one valid digit after the decimal point), and error bars represent standard deviations. Data significance analysis was performed using a t-test (two-tailed, sample-paired method). *: 0.01 < p-value < 0.05; **: 0.001 < p-value < 0.01; ***: p-value < 0.001.

Figure 2

ATP levels associated with the effects of oxidative stress on replication initiation. (A) DNA replication patterns of E. coli. Exponentially growing E. coli cells were cultured to OD₄₅₀ = 0.15–0.2 in ABTG-CAA medium at 37 °C and treated with rifampicin and cephalexin for 3–5 generations (doubling times). Cells were fixed with 70% ethanol and then incubated with Hoechst 33,258 fluorescent dye; 10,000 cells were analyzed for DNA replication patterns by flow cytometry. The number of chromosome equivalents contained per cell is indicated on the X–axis, and the number of cells is shown on the Y–axis. The indicated concentrations of ATP were added at around OD₄₅₀ = 0.04 and co-incubated with the culture for two doubling times to grow to OD₄₅₀ = 0.15–0.2. (B–F) The average number of replication origins contained per bacterial cell in the respective category in panel (A(a–e)). The average number of replication origins per cell (A.O.) represents the sum of the products of the number of chromosomes and the percentage of related cells. Data are the average of three independent biological replicates (marked above the corresponding column plot and showing one valid digit after the decimal point), and error bars represent standard deviations. Data significance analysis was performed using a t-test (two-tailed, sample-paired method). *: 0.01 < p-value < 0.05; **: 0.001 < p-value < 0.01.

Figure 3

Proteomic screen and genetic validation of common factors affecting replication initiation during oxidative stress. (A) Global protein expression profiles. H₂O₂, 0.1 mM H₂O₂. WT_control, WT without H₂O₂ treatment. (B) Venn diagram showing the number of proteins that were co-significantly differentially expressed in the various treatments compared to the control. The comparison groups represented by the Arabic numerals correspond to panel (C). (C) Specific names and expression trends of proteins listed in panel (B). UC indicates uncharacterized proteins. ctrl, control (WT without H₂O₂ treatment); Up, up-regulated; Down, down-regulated. (D,F) Effects of the absence of proteomics-screened proteins and their associated proteins on replication initiation under H₂O₂ stress. The average number of replication origins per cell (A.O.) represents the sum of the products of the number of chromosomes and the percentage of related cells. The indicated concentrations of H₂O₂ were added at around OD₄₅₀ = 0.08 and co-incubated with the culture for one doubling time to grow to OD₄₅₀ = 0.15–0.2. (E) Effects of exogenous glycine, glutamate, ornithine, and aspartate on BW25113 (WT) replication initiation. 10 mM glycine (Gly), glutamate (Glu), ornithine (Orn), and aspartate (Asp) were added at around OD₄₅₀ = 0.04 and co-incubated with the culture for two doubling times to grow to OD₄₅₀ = 0.15–0.2. Data are the average of three independent biological replicates (marked above the corresponding column plot and showing one valid digit after the decimal point), and error bars represent standard deviations. Data significance analysis was performed using a t-test (two-tailed, sample-paired method). *: 0.01 < p-value < 0.05; **: 0.001 < p-value < 0.01. Treatments with insignificant differences are not indicated.