Functional Analysis Of A Global Radiation-resistant Regulator IrrE In Salt-stress Tolerance Of Escherichia Coli And Its RpoS Mutant

Deinococcus radiodurance is a member of the class of radiation-resistant microbes. It is extremely resistant to ionizing or ultraviolet radiation, oxidants, desiccation, and genotoxic agents. IrrE protein from D. radiodurance, called the general switch in response to DNA damage, maximally increases the expression of RecA and PprA which are key enzymes in DNA repair system. The sequences and function of IrrE proteins from different members of the Genus Deinococcus are highly conserved. This protein also appears to be part of a putative signal transduction pathway in response to DNA damage. In addition, it is shown that IrrE protein can enhance multiple abiotic stress resistances in Escherichia coli and survival of Brassica napus grown in a high-salt environment. To provide a theoretical basis for future applications of the irrE gene in genetically modified plants, we continuously investigated the function of IrrE protein and examined its regulatory mechanism in the IrrE-expressing strain on the basis of previous research.We investigated the change of intracellular osmoprotectants by HPLC assay and AAS respectively. The results showed that the intracellular content of trehalose and glycerol increased significantly after salt shock in the IrrE-expressing strain, whereas the change of two other osmoprotectants, potassium and betaine, was not significant compared with the control strain.The expression of functional genes and regulatory genes involved in response to osmotic shock was examined by real-time Quantitative-PCR. The results revealed that under normal or salt shock conditions the genes coding for the main functional proteins involved in trehalose synthesis, otsA and otsB, were up-regulated, but both trehalose and glycerol degradation genes were down-regulated. Additionally, some genes involved in RpoS proteolysis, curli synthesis, and acid-resistance were changed significantly, suggesting that IrrE could have multiple regulatory effects. Specially, the expression level of RpoS was increased in the IrrE-expressing strain, suggesting it could probably be involved in IrrE regulation.RpoS positively controls approximately 10% of the genes in the E. coli genome and most of them are related to abiotic stress-resistance. With the expression of the IrrE protein in the rpoS mutant, confirmed by western blot, we investigated the effects of RpoS in the IrrE-expressing strain during abiotic stress. The results showed that the survival of the IrrE-expressing strain was identical to that of the control strain after hot shock, salt shock, and oxidative shock when the rpoS gene was deleted. The growth curve in LB medium supplemented with 0.25% NaCl and Biolog assay demonstrated that the expression of IrrE in the rpoS mutant did not change the cell's growth and metabolism significantly. Therefore, we propose that RpoS could be an important factor in relation to IrrE regulation, but understanding its precise role in regulation awaits further studies. These conclusions could provide some suggestions and a basis for futher studies.