The Cr(?) Resistant Mechanisms Research Of Sporosarcina Saromensis M52 And The Construction Of ChrA Recombinant Strain

[Objective]Chromium(Cr)is one kind of common heavy metal widely used in the industries of electroplating,metallurgy and tanning etc.,which results in the Cr-containing pollutants entering the environmental medium.Theses pollutants not only cause the destruction of ecological environment,but also ultimately threat the survival of human beings through bio-enrichment and biomagnification effects.Hexavalent chromium(Cr(?))and Trivalent chromium(Cr(?))are the main forms of Cr under natural conditions.Between them,Cr(?)has strong biotoxicity and can cause carcinogenic,teratogenic and mutagenic effects.It was classified as Class A pollutant by USEPA.The main treatment methods of environmental Cr(?)include:physical,chemical and bioremediation methods.Bioremediation by microorganisms provides a potentially practical application for its characteristics of cost-friendly,operation-friendly and high efficiency.But microorganisms are vulnerable to the environmental factors,which would make it difficult to ensure the treatment efficiency.Thus,the molecular mechanisms of Cr(?)resistance of the strain Sporosarcina saromensis M52 was to study through multi-omics joint analyses in this research.On the basis of Cr(?)resistant mechanisms,high-efficiency Cr(?)reducing engineering strain was constructed through the gene engineering methods for the purpose of providing the scientific basis for the bioremediation of Cr(?)pollution.[Method]This study was based on the Cr(?)-resistant strain Sporosarcina saromensis M52,which was previously isolated from the sediment samples of intertidal belt in coastal sea of Xiamen,Fujian province.The genome DNA was extracted to amplify the sequence of 16s rRNA for verifying the strain information and the genomics analysis would be performed.The high Cr(?)concentration group contained 400mg/L Cr(?),and the low Cr(?)concentration group contained 50mg/L Cr(?)according to the range of 8 folds.Strain M52 was cultured to the later period of logarithmic phase and the total proteins were extracted to perform the proteomics analysis.According to the results of multi-omics joint analyses,functional genes involved in Cr(?)resistance were screened out to clarify its resistance mechanisms.The gene chrA coding chromate transporter and the genome DNA of strain M52 were used as target gene and the template to amplify the sequence of chrA.Then,the gene chrA was linked with vector pET-28a(+)and transferred into E.coli Trans5? to structure the recombinant expression plasmid of pET-28a(+)-chr A.After that the chrA engineering strain was constructed by transferred the recombinant expression plasmid into E.coli BL2 1 to construct.Last,the Cr(?)removal characteristics of chrA engineering strain was determined.[Result]The 16s rRNA was 1423bp,and the sequencing result was consistent with raw information.The genomics data showed that there were 10 genes participating in Cr(VI)resistance.To be more specific,gene ort3458 coded chromate transporter,which was able to reduce the intake of Cr(VI)by exporting intracellular Cr(VI).The genes orf0423 and ort2987 coded NAD(P)H-dependent FMN reductases,which utilized NAD(P)H and Cr(VI)as electron donor and acceptor to reduce Cr(VI).The genes of or0f415.ort301 5 and ort3237 coded nitrate reductase.NAD(P)H-dependent nitrate reductase and FMN reductase respectively,which were also able to reduce Cr(VI).The proteins coded by g'enes orf1201.orf1202.orf1273 and orfl686 were able to repair DNA mismatch.which was caused by ROS produced in the process of Cr(VI)reduction.The proteomics analysis data showed that the expression of orf503,orf3504,orf3505 and orf3506,which coded ABC transporter,were significantly upregulated to export Cr(?)from the cytoplasm in low Cr(?)concentration group.The proteins related to flagellum synthesis(FlaG?FliS and FlgM)were significantly upregulated to enhance the motility of strain M52 for the purpose of eluding lethal effect caused by high concentration of Cr(VI).And the gene orfl693 related to Tat protein transporting pathway was significantly upregulated,which assisted in the correct synthesis of proteins.Through the gene engineering techniques,the 1194bp of gene chrA that coded Cr(VI)transporter was successfully amplificated.Then the chrA engineering strain was constructed by transferred recombinant plasmid of pET-28a(+)-chr A into E.coli BL21.A 48kD protein expressed in inclusion body was expressed through the induction of 1PTG.It has been verified that the chr.A engineering strain has Cr(?)removal ability,and the 100mg/L Cr(?)could be almost completely removed within 72h.but the Cr(VI)removal ability was slightly lower than that of strain M52.[Conclusion]The multi-omics joint analysis showed that the main mechanisms of Cr(?)resistance involving:The encoding of Cr(VI)reductases to reduce the cy'toplasmic Cr(VI)concentration;Enhancing the Cr(VI)active efflux to decrease the direct damage to the cells;Expressing proteins with DNA repair abilities to reduce the oxidative damage of ROS to DNA;Upregulating the expression of genes related to flagellum synthesis to increase cell mobility to avoid high concentration of Cr(?);Activating Tat pathway to guarantee the correctly proteins synthesis and increasing quorum sensing.The chrA engineering strain was able to remove Cr(?),which laid a theoretical foundation for the construction of other functional gene recombinant strains and showed good practical application potential.