Studies Of Isolation,Detection Of Mercury-resistant Strain And Structural And Functional Of Mercury-resistant Protein

With the continuous development of modern industry,mercury pollution in the environment is becoming more and more serious,which not only destroys the ecological environment,but also threatens human health.How to eliminate mercury pollution problems of the environment poses a difficulty and become a hotspot of research in the world today.Known as "Mercury Capital",Guizhou Wanshan once was the largest mercury mining base in China,where the surrondings are severely contaminated by mercury mining.In order to understand the impact of mercury mining on the surrounding environment,we isolated microbes resistant to mercury from the mercury-contaminated soil sediment from Wanshan area.According the 16S rDNA sequencing method and PCR amplification method,a mercury-resistant microorganism?Hg401?was identified,belonging to the genius of Bacillus.In nature,ecological cycle of mercury-resistant microorganisms in mercury has very important significance,because it plays an important role in the degradation of methyl mercury and mercury ion was reduced to its elements.To understand the physiological characteristics of mercury-resistant microorganism,five different heavy metals?HgCl₂,ZnSO₄,CuSO₄,CdSO₄,CH₃HgCl?for the mercury resistant microbial growth was added in media for its growth.We found that Hg401 strain is more resistant to both HgCl₂ and CH₃HgCl with 0.2 mM and 1.5 ppm,respectively,compared to Escherichia coli.And Hg401 strains also showed a certain tolerance to ZnSO₄ and CdS04.Through further research,we found that the Hg401 strain harbors mer operon,which is the most widely distributed gene cluster responsible for mercury detoxification.Genes in mer operon are classified into three grousps,as for regulater of merR,for transportor of merP and merT,and for inorganic mercury and organic mercury compounds in the bacterial enzymatic reaction detoxification of merB?organomercurial lyase enzyme?and merA?mercury ion reductase?.Hg401 strain,contains one merA and three merB genes encoding organomercurial lyase enzyme MerB1 MerB2 and MerB3 respectively.By the method of polymerase chain reaction?PCR?and molecular cloning method,we constructed recombinant E.coli strains harboring merA,individual merB,and mer operon,respectively,examined their growth in the presence of various metals?HgCl₂,ZnSO₄,CuSO₄,CdSO₄,CH₃HgCl?.Like mercury-resistant strains Hg401,we found that Escherichia coli strains containing mer operon or merA are more tolerant to HgCl₂?0.1 MM?.When merA overexpressed,tolerance levels to HgCl₂ can reach even higher,comparable to Hg401 bacteria?0.2 mM?.In addition,Escherichia coli containing merB 1 or mer operon are more insensitive to CH₃HgCl with 1.5 ppm.In the regard with ZnSO₄ sensitivies,only Escherichia coli including merA gene showed a better tolerance ability.There are three species of MerB homologous proteins in Hg401 strain,their specific functions are not yet known,the related enzymatic catalysis mechanism is still unknown,using structural biology and other methods to analysis the structural and functional of mercury protein MerB,can provide a theoretical reference for mercury-resistant bacteria resistance mechanism and practical application.In this thesis,we have purified recombinant MerB1 and MerB3 proteins overexpressed in Escherichia coli for crystallography study.We found that the MerB1 protein in solution might exist in two states,namely monomer and dimer.In the presence of Hg2.MerB1 forms additional form in solution except monomeric and dimeric MerB1 forms.The additional MerB1 form may represent a fraction of MerB1 bound with mercury with a noval conformation after MerB1-mediated reaction.