Bioengineering radioresistance by overproduction of RPA, a mammalian-type single-stranded DNA-binding protein, in a halophilic archaeon

Abstract

Halobacterium sp. NRC-1 is a wild-type extremophilic microbe that is naturally tolerant to high levels of ionizing radiation. Mutants of strain NRC-1 with even higher levels of resistance to ionizing radiation, named RAD, were previously isolated after selecting survival to extremely high doses of ionizing radiation. These RAD mutants displayed higher transcription levels for the rfa3 operon, coding two subunits of the RPA-like putative single-stranded binding protein, rfa3 and rfa8, and a third downstream gene, ral. In order to bioengineer cells with increased tolerance to ionizing radiation and further explore the genetic basis of the RAD phenotype, we placed the rfa3 operon under control of the gvpA promoter in a Halobacterium expression plasmid, pDRK1. When pDRK1 was introduced into the wild-type NRC-1 strain, overproduction of the Rfa3 and Rfa8 proteins was observed by Western blotting and proteomic analysis. The Halobacterium strains expressing Rfa3 and Rfa8 also displayed improved survival after exposure to ionizing radiation, similar to the RAD mutants, when compared to wild-type strain NRC-1. The Rfa3 and Rfa8 proteins co-purified by affinity chromatography on single-stranded DNA cellulose columns, confirming the ability of the proteins to bind to single-stranded DNA as well as their relative abundance in the wild-type, RAD mutants, and rfa3 operon overexpression strains. These results clearly establish that overexpression of haloarchaeal RPA promotes ionizing radiation resistance in Halobacterium sp. NRC-1 and that the Rfa3 and Rfa8 subunits bind single-stranded DNA. Bioengineering cells with increased levels of ionizing radiation resistance may have potential value in medical and environmental applications.

Fig. 1

The rfa3 operon region. Sequence of the promoter region is shown above, with location of mRNA start site marked by an arrow and putative TATA-box labeled. Below, boxes indicate the relative location and sizes of the rfa3, rfa8, and ral genes.

Fig. 2

Map of Halobacterium sp. NRC-1 expression vector pDRK1. Relative size, location, and transcriptional orientation of bla gene (coding β-lactamase for ampicillin resistance), mev gene (coding HMG-CoA reductase for mevinolin resistance), rfa3 operon, and rep gene (coding Halobacterium pGRB1 replicase) are shown with arrows. Position of the gvpA promoter is indicated by an arrow and labeled P. Locations of KpnI, NdeI and BamHI restriction sites used for construction are indicated.

Fig. 3

Western blotting analysis of Halobacterium sp. NRC-1 and derivative strains using rabbit Rfa3 antiserum. A. Equal quantities of total cell lysate protein samples were electrophoresed on a 12% polyacrylamide-SDS gel, transferred to PVDF membrane, and probed with Rfa3 antibody followed by a secondary goat anti-rabbit antibody-alkaline phosphatase conjugate and chromogenic substrate. Lane 1: Halobacterium sp. NRC-1, Lane 2: Halobacterium sp. LH5, Lane 3: Halobacterium sp. LH7a, Lane 4: Halobacterium sp. NRC-1 (pDRK1). B. Relative intensities of the bands analyzed by ImageJ software. Vertical axis shows the relative band intensity for the same strains as in part A in the horizontal axis. Values are the average of triplicate experiments with standard deviation shown with error bars.

Fig. 4

Surviving fraction of Halobacterium sp. NRC-1 and derivative strains after irradiation with ionizing radiation. Survival curves are shown for Halobacterium sp. NRC-1 (dashed ◆), LH5 (round dots, •), LH7a (square dots, ■), and pDRK1 (solid, ▲) exposed to different doses (0, 1, 2 and 3 kGy) of ionizing radiation (percent surviving in log scale (vertical axis) versus radiation dose in kGy(horizontal axis). Values are the average of triplicate experiments with standard deviation shown with error bars.

Fig. 5

Affinity purification and Western analysis of Rfa3 and Rfa8 in Halobacterium sp. NRC-1 and derivative strains. Total protein samples from cell lysates and fractions binding to a DNA-cellulose column were electrophoresed on a 12% polyacrylamide-SDS gel, and stained with Coomassie blue, or transferred to PVDF membrane, and probed with rabbit Rfa3 antibody followed by a secondary goat anti-rabbit antibody-alkaline phosphatase conjugate and substrate. Panel A contains Halobacterium sp. NRC-1, panel B contains Halobacterium sp. LH5, panel C contains Halobacterium sp. LH7a, and panel D contains Halobacterium sp. NRC-1 (pDRK1). Lanes labeled 1 contain total cell lysate and lanes labeled 2 contain peak bound fraction from a DNA-cellulose column, Coomassie stained, and lanes labeled 3 display results of Western blots.