[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.

[Construction and functional analysis of the crtl gene disruptant in Deinococcus radiodurans].

With the method of Polymerase Chain Reaction and homologous genetic recombination in vivo, the key gene encoding bacterial-type phytoene desaturase (Crtl) which controls the carotenoids biosynthesis pathway in the non-photosynthetic and extremely radioresistant bacterium Deinococcus radiodurans was deleted from the genome. The colorless mutant obtained was designated as M61. Survival rates of mutant strain and wild type strain were investigated under different doses of gamma-radiation and hydrogen peroxide. The results showed that the radioresistant activity of M61 reduced rapidly under ionization radiation, and it became more sensitive to the treatment of hydrogen peroxide especially to high concentration of hydrogen peroxide compared to that of wild type R1. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used to investigate the carotenoid composition of wild type R1 and mutant M61. HPLC results exhibited that the deficient of crtl gene had important effect on pigment biosynthesis pathway, leading to inhibition of the biosynthesis of lycopene and other carotenoids in D. radiodurans. All the results indicated that crtl gene was a key gene controlling the biosynthesis of red carotenoid including lycopene in D. radiodurans. The roles of carotenoids in protecting the bacterial cell from damage by ionization radiation and hydrogen peroxide suggest that the carotenoids contribute to the defense system in D. radiodurans. This study is important for elucidating the radioresistant and antioxidant mechanism in which carotenoids are involved, and it will supply some ideas to the further investigation on the biosynthesis pathway and functions of carotenoids in D. radiodurans.

[Effects of PprI and RecX on antioxidant activity of Deinococcus radiodurans].

Effects of mutations of Pprl (Dr0167) and RecX (Dr1310), which are relative to radioresistance, on reactive oxygen species scavenging activities in Deinococcus radiodurans were investigated using gene mutation, chemiluminescence measurement and enzyme activity analysis. Their possible regulating functions on the activities of antioxidant enzymes was evaluated. Results show that mutant that lacks PprI is remarkably sensitive to reactive oxygen species and its enzyme activities of catalase and superoxide dismutase decrease significantly. On the other hand, RecX has a "negative" effect on reactive oxygen species scavenging activities of this bacterium, i.e., mutation of recX enhances the scavenging activities on reactive oxygen species, and the enzyme activities of catalase and superoxide dismutase in mutant that lacks RecX are significantly increased. These results indicate that these two genes are relative to the regulation of antioxidant system of this bacterium. It presents some idea to the further investigation on the antioxidant mechanism of this bacterium.