Mechanism Of Adaptive Evolution To Irradiation Damage In Escherichia Coli And Proteome Analysis Of Deinococcus Radiodurans Following γ-irradiation

Extremophiles have a number of potential applications,which would benefit from the use of radiation-resistant microorganisms.It begins from the flexibility of microorganisms to adapt to a wide spectrum of extreme environments.Deinococcus radiodurans is one of the most radiation-resistant organisms described to date.Studies show that the resistance is due to D.radiodurans's extremely proficient and accurate DNA repair process.Evolutionary hypothesis about the origin of radiation-resistant microorganisms seems to show a lack of experimental evidence.The origin and evolution of radiation-resistant microorganisms is still remaining unknown in large part.In this study,the founder strain was non-radiation resistant wild-type Escherichia coli K-12.Wild-type E.coli K-12 was exposed to 83 successive rounds ofγ-ray radiation,allowing for full recovery in between each exposure.Strains IR58 and IR21 selected from the final round of irradiated culture showed a dramatic increase in radiation resistance compared to its parental wild-type E.coli strain.In order to understand evolution mechanism during adaptation to irradiation,we applied the whole genome resequencing,comparative proteomics,gene transcription levels and protein oxidation analysis of IR58 and IR21.In order to reveal the mechanisms of extreme radioresistance and DNA repair in D.radiodurans,we also examined intracellular and extracellular proteome changes in D.radiodurans followingγ-irradiation.1.E.coli K12 was forced through cycles of rapid evolution by iterative exposures to doses ofγ-irradiation for 83 cycles.Three robust strains selected from the final round of irradiated culture were named IR58,IR21 and IR51.The three strains showed a dramatic increase in radiation resistance compared to its parental wild-type E.coli strain.IR58 and IR21 also showed a moderately resistant to UV and more resistant to mitomycin C.2.The whole genome of IR58 and IR21 were resequenced by the high-throughput Solexa Genome Anazylers sequencing system.Our comparative study revealed that IR58 and IR21 underwent numerous events including genetic mutation,gene duplication rearrangement and segment deletion during irradiation adaptive evolution.We found differentiation sequences had undergone many non-functionalization events in both IR58 and IR21,and also gene duplication. Numerous single nucleotide polymorphisms(SNPs)were dispersedly exist in whole genome of both IR58 and IR21.We also found that five genes(uvrB.mutM.lexA. hepA and danX)in IR58 and six genes(recC.recJ.dnaE.mutM.hepA and mutS)in IR21 occurred base substitution,insetion or deletion.Based on comparative genomic data,we propose that the whole genome of both IR58 and IR21 suffered numerous events during irradiation adaptive evolution,such as point mutations,gene duplication rearrangement and segment deletion.3.We developed the ProteomeLab PF-2D fractionation system to carry out large scales protein expression analysis of cell lysates from IR58,IR21 and K-12. Compared with wild-type K-12,expression level of 144 proteins had significantly changed in both IR58 and IR21.But 38 proteins were changed in different trends in IR58 and IR21.39 of 144 proteins and 15 of 38 proteins were identified using matrix-assisted laser desorption ionisation time of flight mass spectrometry after tryptic digestion.These proteins exhibited various cellular functions,and four of them were reported to relate to DNA repair.4.The transcription levels of genes related to DNA repair and replication were analyzed in IR58 and IR21.Compared with E.coli K-12,transcription levels of many important genes in IR58 and IR21 were increased,such as uvrABC,recA,ssb,recBCD and recFOR.Protein oxidative injury was analyzed using the 2,4-dinitrophenylhydrazine(DNPH)procedure.Afterγ-irradiation,IR58 and IR21 had lower protein carbonyl content compared with E.coli K-12.The results indicated that the ability to protect proteins in IR58 and IR21 might be well developed,thus becoming a survival factor with irradiation.5.In order to reveal the mechanisms of extreme radioresistance and DNA repair in D.radiodurans,we examined intracellular and extracellular proteome changes following T-irradiation using 2-D PAGE and silver staining.Among the identified proteins,most of them have not previously been reported to be relevant to radioresistance.Five proteins in intracellular proteome were classified in signal transduction and regulation,and two proteins(SSB,PprA)were reported to have concern to DNA repair.Seven in extracellular proteome are classified in transport and metabolism,and one is involved in intracellular trafficking and secretion.