Role Of DrLEA3 In The Extreme Resistance Of Deinococcus Radiodurans

Deinococcus radiodurans,which is well known for its extraordinary radiation tolerance,is able to survive under extreme conditions,including desiccation,salinity and oxidative stress.Based on the similarities between biological response to radiation and drought stress,it is believed that the radiation resistance of D.radiodurans may be due to its adaptation to desiccation.In this paper,we focused on the drought-resistance-related gene dr₁172 and its encoded protein DrLEA3 in D.radiodurans.We investigated the roles of dr₁172 in D.radiodurans,including its classification,functions in vivo,and in vitro biochemical properties?such as maintaining the stability of cell membrane,protecting protein and enzyme activity,metal ion binding and antioxidant,etc.?.The main results are as follows:1.Bioinformatics-analysis indicated that DrLEA3 is a typical group 3 of the LEA family proteins.DrLEA3 is present in most Deinococcus-species and contains a highly conserved N-terminal domain and a highly hydrophilic C-terminal region,which contains several 11-amino-acid repeating motifs.Positions 6,7 and 8 of these repeats are strictly conserved?Lys-Asp-Lys?,and the rest positions have a high frequency of specific amino acids such as Ala,Val and Asp,indicating that DrLEA3 is not strictly conserved.2.By measurements of the growth curve and the phenotypes of the wild-type and dr₁172 mutant strain under hydrogen peroxide stress,we found that the depletion of dr₁172 led to a delayed cell growth and decreased tolerance to oxidative stress,but not sensitive to UV radiation treatments.The results of real-time quantitative PCR showed that the expression of dr₁172 was upregulated under the high salt conditions and high dose of hydrogen peroxide stress.However,the transcriptional levels of dr₁172 did not alter significantly under UV radiation,indicating that DrLEA3 protein was likely involved in the cellular antioxidant processes.The total antioxidant enzymatic activity of wild-type and dr₁172 mutant cells,the transcriptional level and enzymatic activities of three major reductases?SodA,KatE,KatA?were also measured.The total antioxidant capacity of the dr₁172 mutant cells,thetranscriptional level and enzymatic activity of SodA were both decreased compared with that of wild-type cells.Interestingly,the transcriptional level and reductase activity of KatE and KatA in dr₁172 mutant were not consistent: the transcriptional down-regulated KatE was accompanied by the increased reductase activity of KatE,while the transcriptional up-regulated KatA had the lower reductase activity of KatA.These results suggested that DrLEA3 may regulate these reductases in a distinct manner.3.The intracellular concentrations of three metal ions?Mn,Fe,Zn?in dr₁172 mutant significantly decreased compared with that in the wild-type cells.The decrease of Mn ion was the largest,which led to the decrease of the intracellular Mn/Fe ratio of the mutant strain compared with the wild-type strain,indicating that DrLEA3 protein may play a role in maintaining the intracellular metal ion homeostasis in D.radiodurans.The purified DrLEA3 protein could interact with these metal ions and undergo secondary structure formation upon metal ion-binding.In addition,subcellular localization showed that DrLEA3 protein was mainly distributed in cytoplasm and cell membrane,indicating that DrLEA3 was possible to maintain intracellular metal ion homeostasis through interactions with cell membrane and related metal ion transporters.In summary,these results suggested that DrLEA3 protein in D.radiodurans is a conserved group3 LEA family protein,which is involved in the cellular response to oxidative stress.Moreover,DrLEA3 protein is mainly located on the cell membrane and able to interact with specific metal ions,maintaining the intracellular metal ion homeostasis.These studies provide clues and new ideas for the study of the extreme resistance,especially the oxidative resistance of D.radiodurans.