Study Of Heterologous Expression Of Small Heat Shock Protein On Stress Response Of Escherichia Coli Under Dual Of Temperature And Heavy Metal

Small heat shock proteins(sHSPs),members of heat shock protein family proteins,are widely distributed in different species.It is capable of protecting the organism from damage under various environmental stresses such as temperature,environmental pollution,resource crisis,biological oxidation,pathogens.Two small heat shock proteins,AgsA(isolated from Salmonella enterica serovar typhimurium)and CeHSP17(isolated from C.elegans),were used in the current study.Through the heterologous expression of small heat shock proteins of AgsA and CeHSP17 in E.Coli(BL21),we investigated the dose-response relationship and intracellular accumulation under the combined stress conditions of temperature elevation and presence of heavy metal in BL21(DE3)+AgsA,BL21(DE3)+CeHSP17 and BL21(DE3).We further explored the protective effects of small molecular heat shock proteins AgsA and CeHSP17 on E.Coli under the combined stress of heavy metals and temperature.In addition,the role of the small heat shock protein AgsA on the intracellular accumulation of heavy metal and cell recovery were further studied.1.Study of E.Coli's response under the combined stress conditions of temperature and heavy metals:The result showed that the small molecule heat shock protein AgsA produced more obvious stress response,protected E.Coli cells and increases the E.Coli's tolerance to heavy metals Zn and Cd at all temperatures tested especially at 42? and 50?.For heavy metals Zn and Cd,the stress response of CeHSP17 was not significant with protection of E.Coli cells only and increase the tolerance of E.Coli to heavy metals of Zn and Cd at 50 only.For the heavy metal Cu and Pb,the ?small molecule heat shock protein AgsA did not show significant stress response.The protection of E.Coli cells and increase in the tolerance to heavy metals Zn and Cd was only observed at 50?.The protective effect of the small heat shock protein CeHSP17 against heavy metal Cu and Pb was not significant at all temperatures tested as compared to the control group.2.The accumulation study of heavy metal with different concentrations in E.Coli at the same treatment time:The accumulation under stress of heavy metals Zn and Cd showed that the combined stress of heavy metals and temperature promoted the upregulated expression of small molecule heat shock protein AgsA at 37?,42 and 50? ?,thereby reduced the cell number normalized accumulation of Zn and Cd in E.Coli cells at the same concentration,and increased the tolerance of E.Coli cells to heavy metals Zn and Cd with highest tolerance observed at 42?.For heavy metal Cu,the result showed that at 50?,the combined stress of heavy metal Cu and temperature promoted the upregulated expression of the small molecule heat shock protein AgsA,significantly reduced the cell number normalized accumulation of Cu in E.Coli cells,and increased the tolerance of E.Coli to Cu.For heavy metal Pb,the result showed that there was no differences on the cell number normalized accumulation of Pb among all E.Coli groups at all temperatures tested with some improvement of the tolerance to Pb at 50? only.In terms of cell number normalized accumulation and tolerance,the toxicity of the four heavy metals is ranked as Pb>Cd>Zn>Cu(37?),Pb>Cu>Cd>Zn(42?),Pb>Cd>Zn >Cu(50?).3.The accumulation of heavy metal and recovery study of E.Coli at different treatment times:For Zn,the recovery data of E.coli cells showed that the cell recovery after 4 hours of stress was slightly better than the recovery of cells after 2 hours of stress,while for other heavy metals,the recovery of E.coli cells after 2 hours of stress was slightly better than that of cells after 4 hours of stress.As compared to the control group(BL21(DE3)),the test group(BL21(DE3)+AgsA)showed highest cell recovery efficiency of 84.9% after 4 hours of stress of heavy metal Zn.The cell recovery efficiency were 73.8% and 72.5% for 2 hours of stress of Cu and Cd,respectively.For heavy metal Pb,the recovery efficiency of the test group is slightly lower than that of the control group with the highest cell recovery efficiency of 62.8% after 2 hours of stress.The rank of toxicity of the four heavy metals is Pb>Cd>Cu>Zn.In summary,the results of the current study showed that AgsA showed better protection of E.Coli cells than CeHSP17 under combined stress conditions of heavy metal and temperature.The result can be widely applied in industrial production practice,especially in the use of biotechnology for heavy metal pollution control,pollution ecosystem restoration and many other related aspects.The heterologous expression of AgsA by engineering bacteria can greatly increase the survival temperature of such bacteria and will greatly improve the survival rate of engineering bacteria and the removal efficiency in the treatment of heavy metal pollutions.In addition,the result also showed that the cell number normalized accumulation in E.Coli varied for different heavy metals at the same temperature and concentration,indicating that the cellular of accumulation efficiency depends on the trpe of heavy metals.The cell number normalized accumulation in E.Coli cells at the same inhibition rate indicates the strength of this heavy metal to cytotoxicity.This is of great significance for evaluating the toxicity of heavy metals.And the role of AgsA in the recovery of injured cells cannot be ignored.This study concluded that AgsA significantly improved the recovery efficiency of injured cells.This role can be widely applied in many fields,such as medicine,biochemistry,and other relevant fields where cell recovery from stress is needed.