Classification, Identification And The Mechanism Of Radiation-Resistance Of Thermophilic Bactrrium In Radon Hot Springs

Because of its unique physiological characteristics and great industrial potential value, extremophiles has become a hot topic on new resource development of microrganisms. Radiation-resistant thermophilic bacterium belongs to bipolar microorganisms'category, for its dual characteristics of unique resistance to radiation and thermophilic, it shows a great prospect in agriculture, environment, biomedicine, and even in the defense domain. The microbial community of radon hot springs was studied by PCR-DGGE, and radiation-resistant thermophilic microorganisms were classified and identified by the traditional culture-based methods combining with molecular taxonomy, while investigating the characters and the mechanism of radiation resistance of a radiation-resistant thermophilic bacterium. In this study, the total genomic DNA of radon hot spring water samples was extracted by CTAB method.The results showed that the traditional CTAB method was suitable for extraction of genomic DNA. The V3 region of 16S rDNA was then amplified and the squences used for DGGE analysis. The addition of GC clamp to one of the PCR primers insured that the fragment would remain partial double-strand, and the region screened was in the lowest melting domain. Different DNA samples were analyzed. The optimal level of bacteria DGGE denaturing gradient was 35%～60% with DGGE vertical electrophoresis gel.PCR products of different enrichment ways (11 samples) were electrphoresed by DGGE horizontal gel analysis. The samples were dispersed well in DGGE gel. In the DGGE profiles, there were 12 brighter bands which represent the dominant bacteria. The brighter bands were recovered and the library was established by 16S rDNA sequence analysis. The sequences were compared to known sequences in NCBI, and obtained the entire bacterial community in radon hot springs. It revealed there were 11 strains grouped into five different genera; they were Anoxybacillus, Geobacillus, Ureibacillus, Brevibacillus and Aneurinibacillus.16S rDNA homology of another bacterium was exactly similar with uncultured bacterium, and it may be classified as new species. Samples with different doses of irradiation were DGGE fingerprint analysis which showed only those bands standing for Brevibacillus and Aneurinibacillus disappeared. It indicated that the entire bacterial community in radon hot springs was dominated by radiation-resistant thermophilic genus.After Radon hot spring water sample pretreated with 60Co y-ray radiation by 8kGy, the radiation resistant and thermophilic bacteria were cultured at 55℃,35 strains were obtained in total. We applied two molecular methods to facilitate the process of classification including SDS-PAGE electrophoresis of whole bacterial cell protein and amplification of 16S-23S rDNA spacer region. By combining these two methods, we had isolated 8 strains that belong to different genus. These 8 different strains were further confirmed by sequencing the 16S rDNA and being uploaded to NCBI to compare with other bacteria that had been found before. These results showed strain S1, S9 and T9 had high sequence homology (identity>99%) with Anoxybacillus gonensis NCIMB 13933T (AY122325), Ureibacillus thermosphaericus DSM 10633T (AB210995) and Geobacillus toebii SK-1T (AF326278). Tests on morphological, physiological and biochemical characters, fatty acid compositions, (G+C) mol% contents, and 16S rDNA sequencing were conducted for its taxonomy, strain S1, S9 and T9 may be a new species of Anoxybacillus, Ureibacillus and Geobacillus, respectively.To study the properties of strain T9, we further investigated the mechanism of radiation-resistance of a radiation resistant and thermophilic bacterium. The results showed that strain T9 had a tolerance to UV andγ-ray radiation, as well DNA damaging agents including H2O2 and mitomycin C. It may be related to a unique DNA repair system. Some research showed that it could induce generating NO in vivo under stress, and indicated NO was an important signaling molecule of a series of responses in vivo under induced stress. In this essay, the results suggested that it can induce NO bursting during the UV radiation of strain T9. It indicated that NO may be the induced signaling molecules of radiation-resistant under the irradiation stress. Further studies showed that NO specific scacenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimida zo-line-1-oxy1-3-oxide (cPTIO) could effectively suppress the radiation-resistance; while exogenous NO donor sodium nitroprusside (SNP) could improve the ability of the radiation resistance of strain T9. Therefore, NO indeed involved in mediating the resistance to irradiation of strain T9 and may be the key signaling molecule in the process. It provided an important basis on revealing the mechanism of radiation-resistant of strain T9. Another aspect of the mechanism of radiation-resistance was the antioxidant defense system which formatted in the long-term evolution of strain. The results of this essay showed that NO quencher cPTIO could reduce the antioxidant capacity of strain T9; while exogenous NO donor SNP could well protect the strain which standed against the outside oxidative stress. So, NO may be also the signaling molecule in the antioxidant defense system. However, the specific signal transduction processes remained to be further studied.