Deletion Mutations And Functional Analysis Of Pentose Phosphate Pathway Involved In DNA Damage Repair In Deinococcus Radiodurans

Studies on irradiation-resistant bacteria and their mechanism of DNA damage repair have been received considerable attention from the scientific community. In this paper, we focused on Deinococcus radiodurans CGMCC 1.633, an extremely radiation-resistant bacterium, to test whether pentose phosphate pathway (PPP) is involved in DNA damage repair. To exploit new resouces of radiation-resistant bacteria, we identified a bacterium, Kocuria sp. I-7 with highly radiation-resistance, which was isolated from a sand sample of the Gobi in Xinjiang Autonomous Region, China.PPP plays an important role in the regulation of carbon flux in all cells. Glucose 6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway to convert glucose into ribose-5-phosphate (R-5-P). Transaldolase (TAL) is another critial enzyme. Inactivation of TAL could lead to increased accumulation of R-5-P in cell. Our previous study showed that inactivation of PPP could enhanced the sensitivity to UV light, MMC and H₂O₂ in D. radiodurans, suggesting that PPP facilitated cellular DNA excision repair (NER) and DNA mismatch repair (MMR) in NER and/or MMR mutant backgrounds. In this study, the G6PDH-deficient mutant (Δzwf) and the TAL-deficient mutant (Δdr1337) were construted. TheΔzwf strain grew more slowly (doubling time approximately 2.74 hours) then the wild type strain (doubling time approximately 2.51 hours). While the TAL-deficient did not affect the growth of the cells (doubling time approximately 2.49 hours), but incresed the cellular levels of R-5-P. Analysis of the resistance of the mutants to gamma radiation, UV light, MMC and H₂O₂ showed that theΔzwf strain was obviously sensitive to these DNA damaging agents, and the TAL-deficient slightly enhanced the resistance to these DNA damaging agents. To further understand the effect of the PPP on the resistance to DNA-damaging agents, a complementation experiment was performed in vitro. The intermediate metabolites in PPP, glucono-delta-lactone (GDL), D-ribose or inosine monophosphate (IMP) and uridine monophosphate (UMP) could completely restore or partially restore the resistance of theΔzwf strain to these DNA-damaging agents. Our results suggested that the intermediate metabolites, such as NADPH and R-5-P, the important precursors involved in cell growth and DNA biosynthesis generated in PPP, facilitated the DNA damage repair and the inactivation of TAL resulted in the accumulation of R-5-P to enhance DNA damage repair at a certain extent.Furthermore, we isolated several strains with highly resistance to 10 kGray gamma-irradiation. Of these strains, strain I-7 was selected to sequence its 16S rDNA. The phylogenetic analysis based on the 16S rDNA sequences of bacterial strains showed that strain I-7 is almost identical to those of Kocuria rosea, K. erythromyxa, K. roseus, K. polaris (>99%) and it belongs to the genus Kocuria. The strain was designated as Kocuria sp. I-7. The survival curves of UV light indicated that Kocuria sp. I-7 had a highly capability of DNA repair.