Genome-Wide Screening of Oxidizing Agent Resistance Genes in Escherichia coli

Abstract

The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H₂O₂) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents.

Keywords: AKSA library; genome-wide screening; hydrogen peroxide; hypochlorite; oxidative stress.

Figure 1

Bioinformatical analyses of H₂O₂-resistance genes. The subcellular localization of proteins was obtained from the EcoCyc E. coli Database. The identified proteins were classified into functional categories according to their annotated functions in the UniProt-GOA Database and analyzed by DAVID Bioinformatics Resources. Protein–protein functional interaction networks were analyzed with the STRING database. A high-resolution interaction map is shown in Figure S1.

Figure 2

Bioinformatical analyses of HOCl-resistance genes. The subcellular localization of proteins was obtained from the EcoCyc E. coli Database. The identified proteins were classified into functional categories according to their annotated functions in the UniProt-GOA Database and analyzed by DAVID Bioinformatics Resources. Protein–protein functional interaction networks were analyzed with the STRING database. A high-resolution interaction map is shown in Figure S2.

Figure 3

MICs of selected genes overexpressed in MG1655. MIC determination was performed by varying the concentration of the oxidizing agents, with 1 mM increments from 1 to 20 mM in the LB medium or the M9 minimal medium. The lowest concentration at which bacteria could not grow was recorded as the corresponding MIC. Each strain was tested in three biological replicates. All the differences between MICs of the candidate genes and those of the control were highly significant (p < 0.001).

Figure 4

MICs of selected genes inactivated in BW25113 cells from the Keio collection. MIC determination was performed by varying the concentration of the oxidizing agents, with 1 mM increments from 1 to 10 mM for both H₂O₂ and HOCl and 0.25 mM increments from 0 to 1 mM (if MICs were lower than 1 mM) in LB medium or M9 minimal medium. The lowest concentration at which bacteria could not grow was recorded as the corresponding MIC. Each strain was tested in three biological replicates. Significant differences (p < 0.05) are marked with *, and highly significant differences (p < 0.001) are marked with **.