Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans

Abstract

Deinococcus radiodurans and other members of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. A DNA damage-sensitive and natural transformation-deficient strain generated by chemical mutagenesis (strain rec30) was found to be defective in a gene that has extended homology with recA of Escherichia coli. Upon transformation with a chromosomal DNA fragment that contained this deinococcal recA gene from wild-type (wt) D. radiodurans both DNA damage resistance and full transformation competence were restored in the rec30 mutant. Targeted insertional mutagenesis of the deinococcal recA gene was used to construct a mutant isogenic with the wt. The insertional mutant was phenotypically indistinguishable from strain rec30, indicating that the recA defect alone was responsible for observed phenotypic alterations. For example, in the case of ionizing radiation, the D37 of the wt was about 1.75 Mrad, while the D37 of rec30 and the insertional mutant were both 25 krad, a 70-fold decrease. Evidence is presented that expression of the deinococcal recA gene in E. coli is lethal, suggesting that the mode of interaction of the deinococcal RecA protein with nucleic acids or other cellular proteins differs at least in part from RecA of E. coli.