Construction Of An Efficient Markerless Gene Disruption/Integration System In Acidithiobacillus Thiooxidans And Study On Its Copper Tolerance Mechanisms

Biomining,which has the advantages of low cost,simple operation,high recovery and environment-friendliness,etc.,has been widely applied for metal recovery from low-grade and refractory ores that cannot be economically extracted by conventional methods.During the biomining process,heavy metal ions will be continuously dissolved and accumulated in the bioleaching systems.And high concentrations of toxic heavy metal ions will affect the bioleaching efficiency by inhibiting the growth of bioleaching microorganisms.Therefore,the bioleaching efficiency of copper ores which is the most mineral resources processed by biomining will be unavoidably affected by high concentrations of copper ions in the bioleaching systems.And study on copper resistance mechanisms of bioleaching microorganisms will provide theoretical guidance for construction of engineering strains with steady and high copper tolerance,and finally lay foundations for improving the bioleaching efficiency.Acidithiohacillus thiooxidans,an extremely acidophilic,chemolithoautrophic,y-proteobacterium that can survive in extremely high concentrations of heavy metals and obtain energy from the oxidation of sulfur?S0?and reduced inorganic sulfur compounds?RISCs?.As one of the important bioleaching microorganisms,A thiooxidans has been widely applied for biomining,bioremediation,biodesulfurization,alkaline soil acidification,etc.However,for lack of effective genetic manipulation systems and complete genome sequencing information,study on copper tolerance mechanisms of A.thiooxidans remains blank.This study aims to construct an efficient markerless gene disruption/integration system for A.thiooxidans,and elucidate the copper resistance mechanisms of A.thiooxidans.By expressing the ?-galactosidase gene?lacZ???-glucuronidase gene?gusA??galactokinase?galK?and enhance green fluorescent protein gene?cgfp?in Acidithiobacillus strains,the four reporter gene were compared and selected for gene function studies in Acidithiobacillus strains,which laid a foundation for developing markerless gene disruption systems in Acidithiobacillus strainsAn efficient markerless gene disruption/integration system,which is based on the homologous recombination,yeast homing endonuclease I-SceI and counter-selectable marker,was developed in A.thiooxidans.By enhancing the expression of I-Scel gene,the frequency of the second homologous recombination was significantly improved?0.28±0.048?.And by introducing the counter-selectable marker,the efficiency of screening the mutant strains was also significantly improved.Moreover.by using the established markerless gene disruption/integration system,three gene disruption mutants of A.thiooxidans?AcueO,AcopA and AcusCBA?were successfully constructed,and three exogenous genes?copBAf PTgusA and PcopA-gusA?and two endogenous genes?copAAt and cueZ?were successfully integrated onto the chromosome of A.thiooxidans.A multicopper oxidase coding gene?AthiA1₀10100013518??GcnBank KF998570?was obtained by analyzing the annotation information of the draft genome of A.thiooxidans ATCC 19377.The results of bioinformatic analysis,including homologous alignment,gene function prediction,protein domain alignment,the Tat-pathway signal peptide prediction and subcellular localization prediction,showed that the KF998570 protein may have the similar function with CueO from E.coli.By analyzing the promoter of cueO and comparing the copper tolerance and phenoloxidase activity of ?cueO mutant with the wild type strain,the results showed that?1?the response intensity of P cueO was higher to copper ion than to other heavy metal ions,?2?the AcueO mutant was more sensitive to external copper ions,?3?the phenoloxidase activity was nearly completely lost in AcueO mutant and was restored after complemented with plasmid pJRD215-cueO,indicating that CueO from A thiooxidans possesses the typical phenoloxidase activity as other multicopper oxidases.In conclusion,CueO is suggested to have close relation to copper tolerance in A.thiooxidans.A putative copper-translocating P-type ATPase coding gene?AthiA1₀10100003253??GenBank:WP₀10637963?was also obtained by analyzing the genome annotation information of A.thiooxidans ATCC 19377.By analyzing the subcellular localization of WP 010637963 protein and the alignments of its functional domain with copper-translocating P-type ATPase from other microorganisms,WP₀10637963 was shown to be likely related to copper homeostasis in A.thiooxidans.Then by detecting its transcriptional difference with RT-qPCR and its promoter activity,copA gene showed higher response intensity to copper ion than to other heavy metal ions.Furthermore,the copper tolerance of the wild type,the ?copA mutant,the copA gene overexpression strain A.thiooxidans ATCC 19377?pJRD215-copA?and the complemented strain ?copA?pJRD215-copA?were compared.The copper tolerance was nearly completely lost in the AcopA mutant;however,the copper tolerance was not only restored but also significantly improved after complemented with plasmid pJRD215-copA.The maximum copper concentration tolerated by the ?copA?pJRD215-copA?was 50 mM higher than that by the AcopA mutant.The cellular copper concentrations of the wild type and the copA gene duplicated engineering strain A.thiooxidans copA At was determined and compared,and fewer amount of the cellular copper was detected markedly in the high copper tolerance A.thiooxidans copAAt strain than that in the wild type strain.All the results indicated that CopA plays a pivotal role in the copper tolerance as a copper efflux in A.thiooxidansA gene cluster encoding a MerR family transcriptional regulator?AthiA1₀10100011711??GenBank:WP₀10641489?and a putative copper chaperon?AthiA1₀10100011716??GenBank:WP₀10641491?was found in the genome of A.thiooxidans ATCC 19377.By bioinformatic analysis including function prediction,phylogenetic analysis,functional domain alignment and genetic context analysis,the WP-₀10641489 and WP₀10641491 proteins were preliminarily predicted as a copper-responsive transcriptional regulator?CueR?and a copper chaperon?CueZ?,respectively.Therefore,the transcriptional regulatory effects of CueR on cueO,copA and cueR genes were determined by in vivo promoter activity analysis.And the results revealed that CueR could activate PcueO in the presence of external Cu?II?and Ag?I?and repress itself PcueR,but had no effect on PcopA whether in the presence of external copper and silver ions or not.The activity of PcueR promoter was detected very high and was nearly the same as that of Ptac promoter.The higher response intensity and specificity of PcueR to copper ion than to other heavy metal ions were also detected No ?cueR disruption mutant was screened out after many batches of repetitious experiments,which indicated that CueR might be essential and could not be disrupted in A.thiooxidans.In addition,the PTcueZ fusion fragment integrated engineering strains A.thiooxidans PTcueZ and ?copA::PTcueZ were constructed,and their copper tolerant characteristics were measured and compared with that of the wild type and the AcopA mutant.The slightly higher copper tolerance was detected in A.thiooxidans PTcueZ than that in the wild type strain,but no obvious effect was detected in?copA::PTcueZ compared with the AcopA mutant.The results suggested that CueZ may play a role in binding copper ion and transferring it to CopA in the copper tolerance of A.thiooxidansBesides the above identified cueO,copA,cueR and cueZ gene,some other putative copper resistance related genes were also selected by analyzing the genome annotation information of A.thiooxidans ATCC 19377.Then the transcriptional differences of these genes in A.thiooxidans ATCC 19377 and the ?copA mutant were determined when induced with different concentrations of copper ions.The model of copper resistance mechanisms of A.thiooxidans ATCC 19377 was drawn according to the predicted gene functions,the RT-qPCR results and the experimental results of functional identification of some genesIn this study.a copper biosensor strain A.thiooxidans PAgusA,two high copper tolerance engineering strains A.thiooxidans copAAt and A.thiooxidans copBAf were constructed on the basis of the copper resistance mechanisms o f A.thiooxidans ATCC 19377.The higher bioleaching efficiencies of the two high copper tolerance engineering strains were detected in the presence of 20 mM external copper ions when compared with that of wild type,which indicated that the bioleaching efficiency is in direct proportion to the copper tolerance of bioleaching microorganisms.And the copper biosensor A.thiooxidans PAgusA was also preliminarily explored for application in environmental copper detection.