Identification Of The Genes Related To Cadmium Resistance In Escherichia Coli

Cadmium(Cd)is an nonessential and toxic heavy metal and the chronic exposure to cadmium will threat the health of the organisms.However,even if the ecological conditions become worse,some microbes can evolve according to the changes of the environment.Therefore,the further understanding and cloning cadmium adsorption and resistance genes from microorganisms,simulating the molecular evolution of microbes can not only help us to reveal the cadmium resistance mechanism,but also estabilish a theoretical basis for developing the techniques to remediate the cadmium pollution.In this study,the cadmium resistance to different Escherichia coli strains including BL21(DE3),JM109,DH5?,Top10 and Mach was determined.Among those strains,the E.coli BL21(DE3)exhibited the highest cadmium resistance.Therefore,the strain BL21(DE3)was selected as the research target in this study.The fosmid library of strain BL21(DE3)was constructed and screened in the LB plate containing the cadmium.As a result,four cadmium resistant clones was selected and sequence analyses of four clones indicated that the insertion of each fosmid plasmid contained a fragment located at the lambda phage DE3 region.Several genes in this fragment of BL21(DE3)were overexpressed and the cadmium resistance of the recombinant strains were measured.The results indicated that the gene cap B is the key gene related to the cadmium resistance.In addition,the protein Cap B could bind the cadmium in the E.coli cell.Taken together,these results indicated that the cap B gene confers E.coli BL21(DE3)with increased cadmium resistance by chelating cadmium in the cell.However,cap B is a foreign gene to E.coli.We also expected to screen some endogenous genes related to cadmium in Escherichia coli.Therefore,the method of Genome Replication Engineering Assisted Continous Evolution(GREACE)was used to accelerate of evolutionary rate of genome in E.coli and screen the mutants of E.coli with high resistance to the cadmium.As a result,a mutant(8m M-CRAA)with minimal inhibitor concentration(MIC)value 8 m M was generated,which is about 8-folds higher than that of the E.coli BL21(DE3)wide type strain.The genomes of the E.coli mutants with improved cadmium resistance were re-sequenced with the Illumina sequencing platform.It was found that at least 329 Single nucleotide polymorphisms(SNPs)existed in the mutant strain 8m M-CRAA genome compared to the wild strain.The SNPs of the mutants and RNASeq data induced by the cadmium were used to analyze the genes related to the cadmium resistance.The results of overexpression,knockout and mutations on the genes htp X(encoding heat rock protein)and gor(encoding glutathione reductase)indicated that these two genes also give the positive contribution to the cadmium resistance of E.coli.Then,gene knockout of genes htp X and gor further confirmed that they were related to the cadmium resistance.Site directed mutagenesis of V16 and G249 in gene htp X and gor showed that the mutation of these loci could affect the cadmium resistance of these recombinant strains.In this study,we successfully tamed high cadmium resistant strains in the lab,and found some genes cap B,htp X and gor related to cadmium resistance to Escherichia coli,which is completely different from the cadmium resistant mechanism reported of Znt A(a Zn2+/Cd2+/Pb2+ efflux pump).It deepened our understanding of the mechanism of high cadmium resistance of Escherichia coli,and provided new ideas for further application of cadmium resistant microorganisms.