Study On Zinc/cadmium Resistance Genes In Acidithiobacillus Caldus

The mobilization of metal cations from often almost insoluble ores by biological oxidation and complexation processes is referred to as bioleaching.Acidithiobacillus caldus is an acidophilic,thermophilic and chemoautotrophic bacterium,gram negative,belonging to ?-Proteobacteria,that is separation in the acid mine water.A.caldus has high resistance to Zinc and Cadmium,it plays an important role in the middle and later periods of bioleaching.In Peng Wang thesis,adding Cd2+ to A.caldus MTH04,by transcriptome analysis,found that two genes upregulated significantly,that named orf2634,orJ2635.Wherein,orf2634 appaered fold change 4.21 times,orJ2635 appaered fold change 2.95 fold and predictive orf2634 coding Cd resistance transcriptional regulator CadC,orf2635 coding Cd resistance associated permease.In previously reported,cadCA operon encoding two proteins share a common promoter,cadC encodes a transcription repressor protein CadC,can bind Zn2+/Pb2+/Cd2+;and cadA coding Cd efflux ATPase.This operon granted Staphylococcus aureus Cd resistance.Although A.caldus MTH04 encoding these two proteins differences from Staphylococcus aureus cadCA operon,but transcriptome analysis showed that they may be participate in Cd2+ resistance adjustment.Therefore,in this study,we research the function of the gene orf2634 and orf2635,try to explore their function in A.caldus Zn/Cd tolerance.First of all,A.caldus MTH-04 was cultivated under the normal condition,we extracted RNA and reverse into cDNA and analysis whether they have a common promoter.Through the analysis,we found that orf2634 and orf263 5 shared a common promoter,they were cotranscription.In order to further study the role of this operon in A.caldus MTH-04 resistance to Zn/Cd,we knocked out of the gene cluster,and the determinated growth curve of mutant strains under the different concentrations of Zn/Cd,the results shows that the gene cluster knockout cause A.caldus resistance of Zn decline,Cd resistance maybe increase.Secondly,to study the function of orf2635,we knocked out orf2635 in A.caldus and build a deletion mutant(?orf2635);over expression orf2635 in A.caldus and build a overexperssion mutant(pJRD215-Ptac-orf2635).By determinated growth curve of mutant strains under the different concentrations of Zn/Cd,we found that orf2635 knockout cause A.caldus Zn resistance increase,Cd resistance decline.Using RT-PCR analysis,we found that when A.caldus(?orf2635)grown under 0.09 M Zn2+after 9 days,Zn/Cd couldn't induce the fold change of orf2634 transcription;When adding Cd2+ stimulation,causing gene encoding a phosphate transporter protein pstA upregulation,which may be involved in the A.caldus Cd2+ resistance adjustment.Finally,in order to study the regulated mechanisms of orf2634,protein was expression and purification in E.coli BL21.Using EMSA technology,we try to explore the interactions between DNA and protein.metal and protein.As experiment confirmed,orf2634 encoded protein can be combined with the gene cluster promoter DNA,and Cd2 + add this combination can cause dissociation.This article identifies two genes in A.caldus MTH-04 involved in Zn/Cd resistance adjustment,namely orf2634,orf2635;and the two genes within the same operon,orf2634 encode transcriptional regulatory proteins can bind to the operon promoter region,and this phenomenon can slow down after addition of Cd2+ to DNA-Protein compound.