Study On Genes Of Carotenoid Biosynthetic Pathway In Deinococcus Radiodurans R1 And The Identification Of Two New Species

Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, showed high resistance to ionizing radiation, ultraviolet light, desiccation and many chemical agents. The high resistance of Deinococcus radiodurans R1 was attributed to the special cell structure, the scavenging ability of reactive oxygen species and efficient repair capability for DNA damage. Many carotenoids are the natural antioxidants. Deinococcus radiodurans R1 contains a major carotenoid namely deinoxanthin. Its possible antioxidant role in this strain has not been completely elucidated.In this study, two colorless mutants were constructed by knockout of crtB and crtl genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that free radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels within the cells. We further showed that the carotenoid extract from the wild type could obviously scavenge the free radical signals generated by the irradiated crtB mutant cells. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers. To define which type of free radicals was generated in the process of UV radiation of crtB mutant cells, D-mannitol, a commonly used hydroxyl radical scavenger, was added to the mixture before radiation. The presence of D-mannitol resulted in the evident decrease of the free radical signals, suggesting that the free radical signals generated in UV-irradiated crtB mutant cells were mainly hydroxyl radicals. Furthermore, when catalase was added to the mixture, the major signals of free radicals also disappeared. This result indicated that hydrogen peroxide production was a prerequisite to the generation of hydroxyl radicals in the process of UV radiation of crtB mutant cells. The results also demonstrated that carotenoid extract from the wild type could scavenge the free radical signals generated in UV-irradiated crtB mutant cells, and no obvious free radicals were generated in the UV-irradiated wild type. Thus, carotenoids of D. radiodurans R1 act as hydroxyl radical scavengers in vivo. The ESR assay in vitro indicated that carotenoids from D. radiodurans R1 were also capable of scavenging superoxide anion. Therefore, carotenoids of D. radiodurans R1 play an important role in the defense mechanism of this organism by scavenge free radicals including hydroxyl radicals and superoxide anion.Deinoxanthin, the major pigment in D. radiodurans R1, has particular structure which has not been found in other organisms. Thus, the genes in the carotenoid biosynthetic pathway should be much different and further study is required. The genes crtLm (DR0801, encoding lycopeneβ-cyclase) and crtO (DR0093, encodingβ-carotene ketolase) were knocked out and two colored mutants were obtained. The carotenoid extracts of these mutants were also tested by HPLC. The expression vectors of crtB, crtl and crtO genes were also constructed. However, onlyβ-carotene ketolase could be expressed in E. coli. The expression of crtB and crtl genes could not be detected in the SDS-PAGE gel electrophoresis.On the other hand,184 bacterial isolates were obtained from soil samples from Xinjiang Province, China. On the basis of morphological characteristics,16S rRNA gene sequences, partial physiological and biochemical features, and whole-cell fatty acid profiles, these isolates belonged to 31 genera within 5 phyla.Several strains of these isolates showed big differences with any recognized bacterial species and probably belonged to new species or new genera. And then three strains were identified by the approaches of polyphasic taxonomy. On the basis of phenotypic characteristics, chemotaxonomic data, phylogenetic analysis, DNA G+C content and DNA-DNA hybridization, strains X14-1T and X19-1 are considered to represent a novel species of the genus Pontibacter, for which the name Pontibacter korlensis sp. nov. is proposed. The type strain is X14-1T. Strain ZLB-3T is considered to represent a novel species of the genus Hymenobacter, for which the name Hymenobacter deserti sp. nov. sp. nov. is proposed.