| [Objective]Chromium(Cr)pollution mainly comes from the discharge of wastewater from industrial activities.Hexavalent chromium(Cr(Ⅵ))and Trivalent chromium(Cr(Ⅲ))are most common in natural environment.Cr(Ⅵ)has strong genotoxicity and can cause teratogenic,carcinogenic and mutagenic effects,which endangers public health seriously.In recent years,with the increase in the detection of Cr(Ⅵ)from environmental media(water,soil)and food,how to remove Cr(Ⅵ)pollution safely,effectively and quickly from the environment has attracted much attention.Although microbial remediation has the advantages of environmental friendliness,high efficiency and simplicity,the application in the environmental field is limited due to the wide variety of microorganisms and the lack of clear remediation mechanisms.This study was based on Cr(Ⅵ)-tolerant strain Sporosarcina saromensis M52,the molecular mechanisms of tolerating/reducing Cr(Ⅵ)were explored through transcriptomics sequencing analysis,and the two Cr(Ⅵ)-reducing genes screened out were used to construct co-expression genetically engineered strain,furthermore,its ability to tolerate/reduce Cr(Ⅵ),the optimal reduction conditions and the mechanism of removing Cr(Ⅵ)were explored,expecting to provide a theoretical basis for the practical application of microbial remediation towards Cr(Ⅵ)pollution.[Method]In this study,the Cr(Ⅵ)-tolerant strain M52 was used as the experimental strain.After the strain information was confirmed,the seed solution was prepared and inoculated into the control group(0 mg/L Cr(Ⅵ)),the low-concentration group(50 mg/L Cr(Ⅵ))and high-concentration group(400 mg/L Cr(Ⅵ))to culture until the late logarithmic growth period respectively,three parallel samples were set in each group.After the cells were centrifuged,the total RNA of M52 was extracted and the cDNA library was constructed,then the Illumina Hiseq transcriptomics sequencing analysis was performed.Orf2987 encoding FMN reductase and Orf0415 encoding nitroreductase were selected as the target genes,ligated with vector pET-30a(+)and transformed into E.coli BL21(DE3)to obtain co-expression genetically engineered strain.SDS-PAGE was carried out to analyze solubility and localization of the protein.Western-Blot further confirmed the successful expression of the target proteins.Under 37℃,pH of 8.0,the co-expression genetically engineered strain 2987&0415,strain M52,the control strain with the empty plasmid,genetically engineered strain 2987 and genetically engineered strain 0415(pre-construction)were respectively cultured in 0,50,100,200,400,800 mg/L Cr(VI)solution to explore their Cr(VI)tolerance and reduction ability.The temperature was set to 25℃,30℃,35℃,40℃,45℃,and pH of 7.0,7.5,8.0,8.5,9.0,each with five levels,the factorial experiment design was used to explore the effects of different temperature and pH on the Cr(VI)reduction efficiency of the strain to obtain the optimal reduction conditions.After culturing in 100 mg/L Cr(VI)for 48 h,the bacterial samples were prepared for SEM-EDX,XPS,TEM tests to analyse the surface morphology,element composition,changes of functional groups and the chromate reduction products location,thus exploring the Cr(VI)tolerance/reduction mechanism of the strain preliminarily.[Results]According to transcriptome sequencing analysis,more and more genes involved were highly expressed in response to Cr(VI)stress with the increase of Cr(VI)concentration.Compared with the control group,there were 633 and 912 differentially expressed genes in the low and high-concentration groups,respectively,of which 342 and 477 genes were up-regulated significantly,291 and 435 genes were down-regulated significantly.The up-regulated expression genes were mainly related to Cr(VI)transport(including ABC transporter family protein,MFS superfamily transporter protein,multi-drug resistance family efflux pump protein,etc.),Cr(VI)reductase(such as nitroreductase and FMN reductase,etc.),thioredoxin,cytochrome family,DNA damage repair system,two-component signal transduction proteins,transcriptional regulators,etc.Orf2987 and Orf0415 co-expression genetically engineered strain was successfully constructed.SDS-PAGE diagram showed that the fusion proteins were mainly presented in the supernatant,and Western-Blot results confirmed that the two target proteins were successfully expressed.With the increase of the initial Cr(VI)concentration,the growth of the strain was inhibited with a certain degree,and the tolerance to Cr(Ⅵ)decreased,the specific comparison of each engineering strain was strain 0415>strain 2987&0415>strain 2987,they all were not as good as strain M52;as time went by,the reduction rate increased gradually,which reached to 87.29%under 50 mg/L Cr(Ⅵ)in 36 h,and exceeded 86%under Cr(Ⅵ)not higher than 100 mg/L within 72 h,but failed to reach 50%under Cr(Ⅵ)higher than 200 mg/L.In summary,the comparison of Cr(Ⅵ)reduction ability of each engineering strain was strain 2987&0415≈strain 2987>strain 0415,all of them were not better than strain M52.The reduction ability of Cr(Ⅵ)by co-expression genetically engineered strain was affected by temperature and pH,which were interacted with each other.The optimal reduction conditions are 35～40℃ and pH of 7.5～8.5.Under Cr(Ⅵ)stress,the co-expression genetically engineered strain changed its morphology,and was mainly based on Cr(Ⅵ)reduction accompanied by a small amount of biosorption.Electrons could be provided by functional groups such as C-C,C-O,carbonyl,hydroxyl and carboxyl,through which Cr(Ⅵ)could be reduced to Cr(Ⅲ)and absorbed on the cell surface of the strain.[Conclusion]Transcriptomics analysis revealed the tolerance/reduction mechanism of strain M52 towards Cr(Ⅵ):multiple transporters and transmembrane efflux pumps were used to achieve Cr(Ⅵ)efflux,ensuring cells’ survival and enhancing the tolerance towards Cr(Ⅵ);intracellular soluble reductases were used to reduce Cr(Ⅵ),and thioredoxin and cytochrome family could act as electron donors;DNA damage repair system was used to maintain the stability of intracellular genetic material.Orf2987 and Orf0415 co-expression genetically engineered strain was successfully constructed and proved to tolerate/reduce Cr(Ⅵ).The optimal reduction conditions are 35～40℃,pH of 7.5～8.5.The surface of the strain has Cr(Ⅵ)reduction and little biosorption ability,providing a technical and theoretical basis for the construction and application of co-expression genetically engineered strain. |
