Knockout Of PprM Affect The Resistence Ability And The Biosynthesis Of Carotenoid In Deinococcus Radiodurans

Background:Deinococcus radiodurans is an orange-pink and non-pathogenic bacteria which is well-known for its outstanding radioresistant on Earth.PprM is a stress response protein and is closely related with resistance ability to ionizing radiation and oxidation agents.Deinoxanthin is a carotenoid in D.radiodurans.Based on the previously experiment,we obtain the pprM mutant strain,during the process of its functional study,we found that the?pprM has a lighter color than the wild type.Objective:Investigating the growth condition of the wild type and the?pprM under different external stresses of oxidation and desiccation.Detecting whether pprM would affect the vitality of antioxidant enzymes in cells.Exploring whether pprM would affect the biosynthesis of deinoxanthin.Measuring the content of the Mn and Fe ions to explore the function of pprM on ion concentration.Analysing whether pprM would regulate the transcriptional level of related genes,and further elaborating the function of pprM in the resistance mechanism of Deinococcus radiodurans.Method:?1?Expose the wild type and the?pprM under different concentration of H₂O₂ and prolonged dry environment to compare the growth condition.?2?Using fluorescent absorbance method to detect the intracellular accumulation of ROS.?3?Extract total protein to detect the hydrogen peroxide and superoxide dismutase enzyme activity.?4?Extract the carotenoid and usespectrophotometerto analyse the absorbance value,use the LC-MS to compare the relative content of deinoxanthin of the two strains.?5?Use QRT-PCR to explore the influence that knockout of pprM exerting on transcriptional level of deinoxanthin biosysthesis related genes and dps gene.?6?Probe the content of Mn and Fe and discrepant ratio of Mn/Fe through ICP-MS.Result:?1?Knockout of pprM lead the D.radiodurans to be more sensitive to different concentration of H₂O₂ and prolonged desiccation condition.?2?The ability of ROS scavenging also decreased in the?pprM.?3?The ctalase activity decreased in the?pprM but superoxide dismutase activity showed no big difference between the two strains.?4?The detection of absorbance value assay and LC-MS results indicate that the?ppr Mhas lower content of carotenoid.?5?The analysis of QRT-PCR indicate that the deinoxanthin biosysthesis related genes and dps gene were down-regulated in the?pprM.?6?The ICP-MS shows that the knockout of pprM results in the content of Mn decreased but Fe increased,which leads to a lower ratio of Mn/Fe.Conclusion:Knockout of ppr M would affect the antioxidant ability and resistance ability to desiccation.PprM influence the biosysthesis of deinoxanthin and affect the transcription level of deinoxanthin synthesis related genes.Knockout of pprM lead to a decrease of Mn/Fe concentration ratio in D.radiodurans.