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.