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.

Gas Vesicle Nanoparticles for Antigen Display

“…The original system employed large plasmids (pFL2, pFM104d, and pSD104) containing the entire GVNP gene cluster and utilized a host strain deleted for all of the gvp genes (SD109) [ 5 , 6 , 15 ]. In the recently developed bioengineering system, a series of smaller, more easily engineered plasmid expression vectors were constructed, containing a fragment of gvp C expressed from the high-level, cold-inducible, gvp A promoter [ 92 , 95 , 96 ]. Foreign sequences were easily incorporated to produce GvpC fusions for display on the GVNP surface.…”

Haloarchaeal Gas Vesicle Nanoparticles Displaying Salmonella Antigens as a Novel Approach to Vaccine Development

“…NRC-1Δ ura 3Δ gvp C deletion strain, constructed using the ura 3-based gene deletion method 54 , showed a partially gas vesicle-deficient phenotype with small, mainly lemon-shaped gas vesicles. We next constructed a gvp C expression vector series using pMC2, an expression plasmid with the cold-inducible csp D2 promoter, and pDRK, an expression plasmid incorporating the higher-level gvpA promoter 55,56 . A series of PCR amplified gvp C gene fragments (C-series) were then inserted into the expression plasmids to test their ability to bind to GVNPs and complement nanoparticle production.…”

Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria

“…They are known to be safe and non-toxic to humans and display many stress tolerant properties, including survival of desiccation, heat, cold, UV, ionizing radiation, and high salinity (~3 - 5 M NaCl) [12,13]. They are genetically engineered through a facile DNA transformation system and well-developed, regulated expression vectors [14-16]. Halobacterium sp.…”