Study On Mercury Resistance And Enrichment Behavior Mechanism Of A Marine Pseudomonas

Pseudomonas alcaligenes A1 can grow in seawater medium in the presence of 120 mg/L Hg²⁺,and its bioaccumulation capacity is about 179.53 mg/g.In order to explore the mechanism of high mercury resistance and high bioaccumulation behavior of marine bacterium A1,a transcriptome analysis was performed.The results showed that there are 772differentially expressing genes in A11 which grew under Hg²⁺stress compared with A10 growing under no Hg²⁺stress.Among them 663 genes are up-regulated,while other 109 are down-regulated.BA_GLEAN₁0002699,BA_GLEAN₁0002700,BA_GLEAN₁0002701 and BA_GLEAN₁0002702 are functional genes with large differential expression relavant to mercury resistance.The blast program comparison discolsed the above four genes are merA,merP,merT,merR genes,which are key functional genes on the mercury operon.These key genes respectively encode Mercury reductase MerA,Mercury periplasmic binding protein MerP,Mercury transporter MerT and Mercury regulatory protein MerR.Among them,both merT and merP gene have three complete duplicate copies,while merA and merR have only one complete duplicate copy.Moreover,the relative expression levels of merT,merP and merA at different levels of mercury ions were detected by real-time fluorescence quantitative reverse PCR,and the result showed that the relative expression levels of merP is the highest,followed by merT and merA.In order to further study the mechanism of mercury resistance and bioaccumulation behavior of marine bacterium A1 on the cell level,we used genetic engineering technology to clone the target genes into pRSFDuet-1 and pACYCDuet-1 plasmid.By transforming the genes into host cells,recombinant bacteria TP,TPA and A were constructed successfully.Compared with the control strain E.coli BL21 which can only grow under no more than 2mg/L Hg²⁺,the recombinant bacteria TP,TPA and A are able to grow in LB medium containing 60,40 and 20 mg/L Hg²⁺,respectively,whereas the lag period is prolonged to varying degree.By studying the bioaccumulation ability of three recombinant strains TP,TPA,A and control group E.coli BL21,the equilibrium bioaccumulation capacities are found to be110.48 mg/g,94.49 mg/g,83.76 mg/g and 82.29mg/g respectively.In summary,Marine bacteria A1 may achieve mercury resistance and enrichment functions mainly through two functional genes,merT and merP.This study may discover a novel mercury resistance and bioaccumulation mechanism for marine microorganism,thus laying an experimental foundation for the bioremediation of heavy metal pollution in marine environment.