[Construction of a dps mutant and its functional analysis in Deinococcus radiodurans].
Dps (DNA protection during starvation) is a member of the iron-binding protein family in prokaryotes. It has been shown previously that Dps possesses ferroxidase activity and the ability to sequester iron that seems to protect DNA from oxidative damage. Based on the method of Polymerase Chain Reaction and homologous genetic recombination in vivo, the gene (DRB0092) encoding a Dps protein homology in the extremely radioresistant bacterium Deinococcus radiodurans was deleted from the wild type strain R1 genome. The obtained mutant was designated as Kdps and further verified by PCR and sequencing. Survival rates of the mutant and wild type strain were investigated after challenged with different doses of hydrogen peroxide (H2O2). Results showed that the survival rate of dps mutant reduced rapidly under the low concentration of H2O2 (< or = 10mmol/L), while the wild type strain showed no sudden decrease. When the H2O2 concentration was higher than 30mmol/L, the difference of the survival rates between the mutant and wild type was more than 50-folds. The result demonstrated that the loss of dps gene in D. radiodurans made cells become more sensitive to oxidative damage. An iron staining method was used to determinate catalase activity in native polyacrylamide electrophoresis gels. The result displayed that two catalases in dps mutant were enhanced about 2-folds than that of wild type. The soluble Dps protein was obtained after construction of expression plasmid and inducement in E. coli transformant. The Dps protein showed the capacity of DNA binding and protected DNA from hydroxyl free radical cleavage in vitro. This study demonstrates that Dps protein of D. radiodurans plays an important role in its antioxidant system, which may contribute to its extreme resistance of this bacterium.
Yan
Zhuo-yan
ZY
Institute of Nuclear-Agricultural Sciences, Zhejiang University, Key Lab of Nuclear-Agricultural Sciences of Ministry of Agriculture, Hangzhou 310029, China. yanzhuoyan@hotmail.com
Xu
Zhen-jian
ZJ
Xu
Guang-zhi
GZ
Tian
Bing
B
Hua
Yue-jin
YJ
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D034301:Deinococcus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009154:Mutation; D018384:Oxidative Stress
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C083911:DPS protein, Bacteria; D006861:Hydrogen Peroxide