The Research On The Function And Mechanism Of Microlunatus Phospho Vorus In Enhanced Biological Phosphorus Removal System

Phosphorus accumulating bacteria play an important role in EBPR,Microlunatus phosphovorus（MLP）as a kind of PAOs has a strong ability to accumulate and release phosphorus.The MLP JN459 which separated and purificated from laboratory was used to research the function and mechanism in the process of removing the Phosphorus.By analyzing the environmental resistance of Microlunatus phosphovorus（MLP）,The minimal inhibitory concentration（MICs）of MLP to most antibiotics was more than 8μg/mL.The MICs to heavy metals is generally maintained at the order of 10^-4 mol/L.When the volume concentration of petroleum hydrocarbons in the environment is less than 1%,the growth of the MLP is not affected.The protein function analysis after the genome sequencing is showed that the antibiotic resistance gene and the heavy metal resistance gene are contained in the MLP.It is an intrinsic reason for its high environmental resistance.Through the research on the phosphorus accumulation capacity of MLP under anaerobic and aerobic conditions,it found that the concentration of soluble phosphorus in the medium increased under anaerobic conditions（about 40 mg/L）,and the concentration decreased under aerobic conditions（about 35 mg/L）.under anaerobic condition,the Poly-P will decreased,while under aerobic condition,the Poly-P increased.Under different dissolved oxygen conditions,real-time PCR technique was used to find that the expression levels of genes MLP₁7420,MLP₂66100,MLP₄4770,MLP₄7700,MLP₄7360 and MLP₅1060showed significant changes in anaerobic conditions.By Using western-blot test method,the research found that the expression of MLP21700 protein was basically the same under different dissolved oxygen levels,but with the growth of MLP and the change of dissolved oxygen conditions,three bands appeared.Therefore,it is speculated that anaerobic can induce a homologous protein of MLP21700.The regulation of MLP21700 protein is mainly affected by phosphorylation rather than expression.the vitro binding research of MLP21700 protein and promoter was carried out by the way of plasmid vector construction,protein expression purification and EMSA,which showed that MLP21700 protein has strong binding ability to promoters MLP00530,MLP26610,MLP44770,MLP47700,MLP47720 and MLP51060.the binding site of MLP21700 protein to MLP0530 and MLP44770 genes was studied by DNase I method.The results showed that the binding site gene sequence to probe MLP0530 was GATAGAGCCGAAAGACCCG,and the sequence of the binding site to probe MLP44770 was GCCCGGGTCCAACGACCCG,a similar binding site was found in the gene sequence comparison,and the conserved binding sites of MLP21700 protein with MLP26610,MLP47700,MLP47720 and MLP51060 genes were predicted.MLP21700 protein have strong binding ability to gene MLP00530,MLP26610,MLP44770,MLP47700,MLP47720,MLP51060 in vitro.By giving the binding site prediction,it provides a reference for the binding of MLP21700 protein to the above genes in the bacteria.