Abstract
Rubrobacter radiotolerans is the most radio-resistant eubacterium without sporulation. Its half lethal dose for gamma-rays is 16,000 Gy. Although it is 2.3 times more resistant for gamma-rays than Deinococcus radiodurans, a well-known radio-resistant bacterium, we have less knowledge about the radio-resistant mechanism of R. radiotolerans. In this study, we analyzed structure of the superoxide dismutase (SOD) and function of its product for elucidation of the radio-resistant mechanism. R. radiotolerans SOD (Rr-SOD) protein was purified by sequential chromatographic procedures and its partial amino acid sequence was determined. Rr-SOD gene and the adjacent genomic regions were mapped by the gene cloning with the partial protein sequence and 5'- and 3'-RACEs. Rr-SOD gene contains 618 nucleotides producing a 206 amino acid-protein containing a manganese as cofactor. Rr-SOD protein shows 68%-homology and 78%-similarity to the ortholog of Rubrobacter xylanophilus. The upstream region of Rr-SOD gene contains the open reading frame of putative PAS/PAC sensor protein like R. xylanophilus. On the other hand, the downstream region lacked any corresponding genes, indicating no similarity between the downstream of the two Rubrobacter species. The whole Rr-SOD gene was inserted in an expression vector for Escherichia coli, and the gene was introduced in the cell of E. coli strain lacking SOD genes. The complemented cells partially recovered its resistance for both gamma-rays and methyl viologen, a compound producing superoxide. Our results indicate that Rr-SOD plays an important role in resistance for ionizing radiation of R. radiotolerans.
