Extremely Radiation-Resistant Mutants of a Halophilic Archaeon with Increased Single-Stranded DNA-Binding Protein (RPA) Gene Expression

Abstract

Extremely halophilic archaea are highly resistant to multiple stressors, including radiation, desiccation and salinity. To study the basis of stress resistance and determine the maximum tolerance to ionizing radiation, we exposed cultures of the model halophile Halobacterium sp. NRC-1 to four cycles of irradiation with high doses of 18-20 MeV electrons. Two independently obtained mutants displayed an LD(50) > 11 kGy, which is higher than the LD(50) of the extremely radiation-resistant bacterium Deinococcus radiodurans. Whole-genome transcriptome analysis comparing the mutants to the parental wild-type strain revealed up-regulation of an operon containing two single-stranded DNA-binding protein (RPA) genes, VNG2160 (rfa3) and VNG2162, and a third gene of unknown function, VNG2163. The putative transcription start site for the rfa3 operon was mapped approximately 40 bp upstream of the ATG start codon, and a classical TATA-box motif was found centered about 25 bp further upstream. We propose that RPA facilitates DNA repair machinery and/or protects repair intermediates to maximize the ionizing radiation resistance of this archaeon.