Study On Enhances Of Stress-resistance Of Bacillus Subtilis By Expression Of Deinococcus Radiodurans PprⅠ

The polyextremophile bacterium Deinococcus radiodurans is characterized by its unusual capability to tolerate numerous DNA damaging agents, including ionizing radiation, ultraviolet radiation, and desiccation. Its unprecedented ability to avoid DNA damage in the presence of heavy irradiation has made it a critical model for pursuing genes involved in maintaining DNA integrity and stability. Studies have shown that the recently identified pprI of Deinococcus radiodurans is crucial factors for DNA damage repair after irradiation. It was confirmed that PprI protein(Deira-PprI), the gene product of pprI, serves as a general switch for downstream DNA repair and protection pathways via its regulatory function on the gene expression of recA, pprA and enhancing the enzyme activities of catalas. Deira-PprI has no high similarity to any previously characterized proteins in NCBI gene bank. RecA or RecA-like proteins have been found throughout the phylogenetic tree, including archaea, bacteria, yeast, plants, and mammals. It plays a critical role in biological processes that require homologous DNA pairing and recombination.RecA of Deinococcus radiodurans and Bacillus subtilis exhibit much identity.So, we hypothesize that expression of this exogenous pprI gene in Bacillus subtilis can turn on a unique regulatory pathway and up-regulate DNA protection and damage repair effectors in response to H2O2 oxidative stress and ultraviolet irradiation.To evaluate whether pprI also function in the resistance in other organisms, we got pprI gene by PCR-coding, the PCR product was ligated into the shuttle vector pRADZ3, which was transformed into Bacillus subtilis, PprI protein was expressed in normal growth condition without induction,empty plasmid pRADZ3 was transformed to Bacillus subtilis as control. The viabilities under H2O2 oxidative stress and ultraviolet irradiation of the two reconstructed Bacillus subtilis were observed.For decades, it has been observed that Deinococcus Radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents. Recently, it was shown that the Deinococcus Radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. For example, studies have shown that the recently identified pprI and recA of Deinococcus Radiodurans are two crucial factors for DNA damage repair following H2O2 oxidative stress and ultraviolet irradiation. Although pprI gene is unique in Deinococcus Radiodurans, so far, no homologues have been identified in other species based on genomic database searches. In contrast to recA is required only for the repair of extreme DNA damage in D. radiodurans and it is expressed only when DNA is severely damaged. Significant induction of recA expression following DNA damage has been observed, recA is a major DNA repair gene whose expression is strictly regulated by pprI. So we hypothesized that recombination expression of the pprI gene in Bacillus subtilis may enhance the bacterium's DNA damage response ability and increase its resistance. Indeed, we were able to demonstrate that the B-1(pprI+) cells showed approximately 2-fold greater survival than B-2 cells at the growth phase, 1.2-fold at the stable phase under H2O2 oxidative stress and 3-fold under ultraviolet irradiation. This indicates that expression of pprI in Bacillus subtilis can enhance the ability to DNA repair by enhancing the enzymatic activity of recA. The increase in activity of recA may contribute to the enhanced resistance in the recA-defective B-1 strain following H2O2 oxidative stress and ultraviolet irradiation. So we know that this exogenous pprI gene can turn on a unique regulatory pathway and up-regulate DNA protection and damage repair effectors in response to H2O2 oxidative stress and ultraviolet irradiation for wild-type cells.In summary, response to DNA damage is mediated by a signal transduction pathway consisting of sensors, transducers, and effectors. DNA damage is detected by sensor proteins, which in turn activate PprI, a major signal transducer. Activated PprI, as a general switch, regulates the expression and activation of a number of DNA repair and protection genes, including recA, pprA, and catalase. Although PprI is unique to D. radiodurans, finding its functional homologues and characterization of their shared mechanism in eukaryotic systems will contribute to the deeper understanding of radiation damage, repair, and resistance, as well as to the improvement of human health.