Functional Characterization Of A Ferritin DrfE Involved In Oxidative Resistance In Deinococcus Radiodurans

Iron is an indispensable element for normal growth of organism,the over-dose iron ion could cause a Fenton reaction generatingROS,which leads to oxidative damage to cells.Some studies have shown that ferritin will work up resistance to oxidative stress and regulation on the concentration of iron ion in cells.Bioinformatics prediction displayed that there is an ferritin-like protein?DRB0118?from Ferritin₂ superfamily in Deinococcus radiodurans,which is extremely resistant to ionizing radiation and oxidants.Research showed that the inactivation of DRB0118 will cause more sensitive to desiccation,but the knowledge of its response to oxidation is unknown..In this study,the function of DrfE in response to oxidative stress was characterized through bioinformatics analysis,mutation and heterologous expression.The main contents and results were as follows:?1?The DrfE protein was analyzed by bioinformatics,the results showed that drfE gene was located on the giant plasmid of the D.radiodurans.DrfE belongs to Ferritin₂ superfamily because it is composed of 337 amino acids and containing a conserved domain,which is an analogous to ferritindesignated DrfE?Deinococcus radiodurans Ferritin,DrfE?.Amino acid sequences analysis showed that DrfE is a typical transmembrane protein containing obvious transmembrane and hydrophobic regions.?2?To investigate the stress resistance function of drfE gene in Deinococcus radiodurans,the drfE deletion mutation strain??drfE?and the complemented strain?pdrfE::??were constructed by fusion PCR and homologous recombination.And compared the stress resistance of the wild-type DR,AdrfE mutant and complemented strain pdrfE::? under different abiotic stresses.The results showed that AdrfE was more sensitive under NaCl and H2O2 stresses,the survival rate is four orders of magnitude lower than the wild-type after 80 Mm H2O2 for 30 min and two orders of magnitude lower after 5M NaCl for 6 h,and drfE gene complementation could restore the resistance defect of the strain.?3?CAT and SOD activities of the wild-type DR,mutant AdrfE and complemented strain pdrfE::A are measured.Under the normal condition,the deletion of drfE resulted in the CAT and SOD activities decrease?CAT activities of the mutant AdrfE decreased by 28.36%and SOD decreased by 6.42%?.Especially under the challenge of H2O2 for 30min,the CAT activities of the mutant AdrfE decreased by 32.33%and SOD decreased by 41.30%.Also,the Fe2+ concentration of the three strains were measured,the result displayed that the deletion of drfE resulted in the Fe2+ concentration increase from 232 ?mol/L to 293?mol/L in vivo.?4?Heterologous expression of DrfE protein in D.radiodurans to E.coli BL21?DE3?,phenotypic analysis showed that DrfE protein can enhanced the stress resistance to NaCl and H2O2 of E.coli.The survival rate of recombinant bacteria increase four orders of magnitude after 15 Mm H2O2 for 10 min and one order of magnitude after 5M NaCl for 2 h.drfE gene in D.radiodurans was involved in salt and antioxidant stress.it can keep the activities of CAT and SOD and increase the Fe2+ concentration effectively,protects the cell from oxidative damages.Molecular mechanism of drfE has to be studied further.