The Role Of Reactive Oxygen Species In Deinococcus Radiodurans' Proliferation And Radioresistance

Deinococcus radiodurans (Dr.) is a bacterium that is extremely resistant to the lethal and mutagenic effects of ionizing radiation. The mechanism of its radioresistance remained unknown. The ionizing radiation can induce Reactive Oxygen Species (ROS) through indirection effect. ROS includes Superoxide (O2), Hydrogen peroxide (H2O2), Hydroxy anion(HO') and Nitrogen monoxide(NO). The effect of different concentration of ROS on Dr. is the question to discuss in this study.Determine the release of O2 ' of Dr. with cytochrome C reduction method, and determine the Dr.'s proliferation with 3H-TdR incorporation method. Then add Diphenyleneiodonium (DPI), the inhibitor of NADPH oxidase, into the culture medium. Determine the release of 02.-. and Dr.'s proliferation in the same circumstance. Measure the concentration of H2O2 in Dr. with Laser Confocal Scanning Microscope (LCSM). It is found that the release of O2 and H2O2 is related with Dr.'s proliferation. DPI can specifically inhibit the release of 02.- and Dr.'s proliferation. Dr's proliferation and the release of 02.- and H2O2 can be stimulated when caused by radiation less than 1KGy and inhibited by high level radiation. The result indicates that the NADPH oxidase exists in Dr. The proliferation of Dr. depends on low level of 02.-. Radiation less than 1KGy can stimulate the proliferation of Dr., the mechanism is related with the release of H2O2 and O2.-. The molecular mechanism of the effect was left for discussion.Irradiate Dr. with different doses of 60Co Y -rays, and determin the activity of superoxide dismutase (SOD) and catalase (CAT) in the bacterium immediately after irradiation. Determined the activity of SOD and CAT in Escherichia coli (Ecoli) strain JM109 as contrast. We found that the activity of SOD and CAT in Dr. were higher than those in Ecoli, and they were increased when Dr. was irradiated by Y -rays. The amplitude of change of the activity of CAT was larger than SOD's. Contrast the gene sequence which encode SOD and CAT in Dr. and Ecoli through BLAST program. It isdiscovered that the homology of gene sequence coding SOD between these two bacteria was 76%, but there is lower homology of gene sequence coding CAT between them. The result indicates that high concentration of ROS can induce the synthesis of SOD and CAT in Dr. This effect was part of the mechanism of Dr.'s radioresistance, and CAT may play a more important role than SOD does.Increase and decrease the activity of CAT in Dr. artificially. Then irradiate it with different doses of 60Co y -rays. Determine the survival rate and DNA damage by colony formation assay and fluorometric assay of DNA unwinding respectively. It is found that when the activity of CAT was increased, the survival rate of Dr. after irradiation was also increased and the DNA damage was significantly reduced. When the activity of CAT was inhibited, the survival rate decreased and DNA damage was more serious. This result indicates that the synthesis of CAT was an important mechanism of Dr.'s self-protect when affected by high concentration of ROS.