The Role Of Peroxidase EfeB In The Regulation Of Oxidative Stress In Escherichia Coli

Oxidative stress is caused by reactive oxygen species?ROS?.ROS are a class of highly chemically active small molecular substances produced by organisms through oxidative metabolism or produced by specific enzymes,including superoxide anions?O₂^-?,hydrogen peroxide?H₂O₂?and hydroxyl radicals?·OH?.Previous studies on microbial oxidative stress have mainly focused on the stress mechanism of the transcriptional regulation of antioxidant systems such as OxyR and SoxRS.However,the regulatory mechanism of peroxidase EfeB in oxidative stress in microorganisms has not been elucidated.In this study,Escherichia coli?E.coli?was taken as the research object,and EfeB was used as the regulatory target.Through the overexpression and deletion of efeB,the effect of exogenous addition of H₂O₂ and?-polylysine??-PL?on intracellular oxidative stress as well as the role of Efe B in it was investigated.The main research contents and results are as follows:?1?Establishment of oxidative stress conditions and improvement of ROS measurement method.The growth curve of the starting strain was measured based on the addition of different concentrations of H₂O₂ or?-PL.And the concentration of 4 mmol·L^-1 H₂O₂ or 55?g·mL^-1?-PL were adopted to cause the oxidative stress which led to significant growth inhibition of Escherichia coli,but not cell death.For the starting strain,laser confocal microscope was used to qualitative detection of intracellular ROS and the best staining conditions for Escherichia coli was determined.Subsequently,flow cytometry was used for quantitative detection of intracellular ROS,which improved the accuracy of the measurement.?2?Regulation of oxidative stress in Escherichia coli by constructing the efeB overexpression strain?the efeB deficient strain and the efeB complementation strain,it was found that over-expression of efeB promoted the growth of the bacterial cell,the content of intracellular malondialdehyde?MDA?decreased 5.66 fold compared to the starting strain?223.79 mmol·mg^-11 protein?,and intracellular ROS is 1.36 fold that of the starting strain.While the catalase gene katE is down-regulated by 8.08 fold,the efeB related genes efeU,efeO and other key genes in the antioxidant system sodA,oxyR,and recA were up-regulated by 3.88 fold,1.33 fold,2.48 fold,1.95 fold and 1.49 respectively.Under H₂O₂ stimulation katE was further reduced by 19.27 fold,and recA was further increased by 8.09 fold in the efeB overexpression strain.Under the stimulation of hydrogen peroxide,the growth of the efeB deficient strain was lower than that of the other two strains.In addition,the content of intracellular MDA of the efeB deficient strain is 1.15 fold that of the starting strain,and the intracellular ROS is 0.5 fold that of the starting strain.?3?Regulation of oxidative stress in Escherichia coli under?-PL stimulation.Using?-PL with a final concentration of 55?g·m L^-11 to stimulate the starting strain,the efeB overexpression strain and the efeB deficient strain,it was found that the growth status of the efeB overexpression strain better than that of the other two strains.Combined with the intracellular MDA content and ROS level of the three strains,it was shown that the overexpression of efeB can play a role in promoting growth under the pressure of oxidative stress,which can reduce intracellular oxidative damage.Compared with H₂O₂ treatment,MDA content of the starting strain increased by 70%,MDA of the efeB deficient strain increased by 78%after treatment with?-PL at a final concentration of 55?g·mL^-1.The MDA content is 4.46 fold in the efeB overexpression strain comared with that of H₂O₂ treatment,indicating that the addition of?-PL will further enhance the level of cell membrane lipid peroxidation,and the intracellular oxidative damage.