The Functional Research Of Two Antioxidant Proteins And Carotenoids Preparation In Deinococcus Radiodurans

Deinococcus radiodurans is one of the powerful species that are extremely resistant to ionizing radiation. It also has extreme resistance to ultraviolet, hydrogen peroxide and other DNA-damaging agents. To combat with these dangerous materials, it develops a series of protection, resistance and repair systems. Previously studies showed its super antioxidant ability contributes to the powerful resistance. This study is to explore the antioxidant mechanism of D. radiodurans for better understanding its resistant ability.The RecG helicase is an important protein being function in the DNA damage repair, which is found to be active under oxidative stress in Pseudomonas aeruginosa recently. Here, we constructed a mutant strain lacking D. radiodurans recG gene, and investigated DRRecG's function. Our results showed that there was a drastic decrease resistance toγ-irradiation and hydrogen peroxide in the drrecG mutant. The ROS scavenging abilities in cell extracts were reduced because of the deficiency of drrecG gene. Further studies on drrecG function demonstrated that the deficiency of drrecG has a negative effect on activitives of antioxidant enzymes in cell extracts and the genome stability under hydrogen peroxide treament. These results suggested that the DRRecG is an important DNA damage repair protein and antioxidant protein under oxidative stress; it may play a role in stabilizing the genome stability and maintaining the activities of catalases. Global transcriptome profiles showed that a lot of genes involved in energy metabolism were up-regulated and the genes coding ribosome proteins were depressed. It is thought that energy metabolism in drrecG mutant was disrupted, consisting with the growth delay in the mutant strain. Furthermore, under the low dose stress of H₂O₂, a lot of DNA repair genes, damage response genes, and anitoxidant genes were highly expressed. These results reflect a serious surviving challenge to D. radiodurans, and illustrate the importance of the drrecG's function under oxidative stress.From our lab's recent results of microarray and 2-DE gel experiments, we found a novel gene dr1127 was up-regulated when R1 strain was treated with 2,000 Gy (sometimes the damage of this dosage is thought to be similar to that of oxidative stress) in D. radiodurans. We successfully constructed the null mutant strain of dr1127 gene, designated as MT1127. Survival test proved that the deficiency of dr1127 did affect the survival ability of D. radiodurans underγ-radiation treatment and H₂O₂-induced oxidative stress. The deficient strain-MT1127 is quite sensitive to the oxidative stress even at a very low concentration of H₂O₂ compared with R1 strain. The free radicals scavenging activities of MT1127 were obviously weaker. The cell extracts of MT1127 exhibited a lower scavenging ability on superoxide anion, hydrogen peroxide than R1, and showed an especially weak ability to scavenge hydroxyl radicals. These results help us to understand why the MT1127 strain is more sensitive toγ-radiation and oxidative stress than wild type strain R1, and suggests that the novel gene dr1127 may have an antioxidant function. The results from oxidative damage assay in vitro demonstrated the DR1127 protein has the function of DNA protection, and its DNA binding activity helps us to further understanding the DR1127 protein's defense function in the oxidative damage. So we can conclude that dr1127 does take part in the antioxidant process, and acts as the protector and guardian of DNA in D. radiodurans under the oxidative stress. As a novel gene, it may be involved in a complex antioxidant system.Carotenoids are the source of red-pigmented D. radiodurans. However, there is no report about fermentation and production of carotenoids from this bacterium up to date. We investigated extraction methods and fermentation conditions for carotenoids production from D. radiodurans. With the optimized medium, the production of carotenoids reaches to 2.318 mg per gram dry mass. Among different organic solvents, extracting production from high to low are methanol, methanol and acetone, aceton, and n-butyl alcohol. As a prokaryote, production of carotenoids from D. radiodurans has advantages of short fermentation period, easier extraction and process control. It will supply with a new strain to the production of carotenoids from microorganism.