Study On The Metabolism Of Nicotinoid Insecticide Acetamiprid And Its Nitrile Metabolizing Enzymes By Plant Rhizosphere Growth-promoting Bacteri

Neonicotinoid insecticide acetamiprid is widely used in the prevention of pests and plant diseases and increasing crop yields.However,in recent years,the extensive and intensive application of ACE caused many environmental and ecological risks.The contaminant soil remediation by plant growth promoting rhizobacteria(PGPR)not only reduced its residue,but also promotes the growth of plant.However,the degradation of ACE by PGPR was investigated rarely.In present study,the author screened two PGPR that could degrade ACE from lab culture collection and further investigated their metabolic pathway and enzymatic mechanism of ACE degradation and explored their IAA production mechanism concerned with PGPR trait.The preserved Ensifer adhaerens CGMCC 6315 and Variovorax boronicumulans CGMCC 4969 from the culture collection all could degrade ACE to amide ACE(IM-1-2).We further compared the two strains with another ACE degradation bacterium E.meliloti CGMCC 7333.The resting cells of CGMCC 7333 showed the highest ACE degradation ability in LB culture condition,and degraded 50.94%of 50 mg/L ACE in 96h.In contrast,after inoculating CGMCC 7333 into soil for 10 d,the ACE degradation rate reached to 28.20%,whreas,the ACE degradation rate by CGMCC 6315 reached to 58.87%at the same condition.After inoculating CGMCC 7333 into water for 72 h,the ACE degradation rate reached to 67.02%,whreas,CGMCC 6315 degraded ACE entirely.Those facts indicated that CGMCC 6315 had higher ACE degradation ability in the soil and water than CGMCC 7333.We further investigated the ACE degradation mechanism by CGMCC 6315 and found that the ACE degradation activity was regulated by nutrient concentration.The ACE degradation efficiency was improved with the decreased nutrient concentration.The resting cells of CGMCC 6315 cultured in LB broth,degraded 17.44%of 200mg/L ACE in 96h,while,the resting cells of CGMCC6315 cultured in 1/15LB broth degraded 94.25%of 200mg/L ACE in 12h and quickly eliminated 87.42%of 5 mg/kg of residual soil ACE within 2 d.CGMCC 6315 degraded ACE to IM-1-2 by nitrile hydratase.The whole genome sequencing analysis showed that CGMCC 6315 contained two NHase-coding genes(cnhA and pnhA).Gene cloning expression and enzyme assay showed that CnhA had a specific activity of 0.46 U/mg toward ACE,whereas,the specific activity of PnhA reached to 28.75 U/mg,which was the highest report about nitrile hydratase activity toward ACE.QPCR and proteomic analysis showed that the improved ACE degradation ability in low nutrient condition was attributed to the up-regulated expression of PnhA.NHase activator protein plays an important role in the mature of NHase,but its expression level was low.We successfully improved the expression level of CGMCC 4969 NHase activator(AnhC)by adding SD sequence and T7 strong promoter in the front of anhC,and adopting double-plasmid expression system.CGMCC 4969 NHase activity was improved by 18.1-fold by co-expression of NHase with AnhC.CGMCC 4969 NHase activity was improved by 21.3-fold when we added the SD sequence upstream of anhC.The expression level of AnhC was further improved,when we adopted the double-plasmid and double-promoter expression system to express the NHase and activator protein,but the NHase activity was reduced.Those facts indicated that the improvement of NHase avtivity requires an optimal expression level of activator protein and the NHase activity was inhibited when the activator protein was excessive expression.Most of the PGPR have the ability to secrete IAA to promote plant growth.CGMCC 4969 cannot directly synthesize the phytohormone IAA via tryptophan,but can produce IAA using indole-3-acetonitrile(IAN)as the precursor.The whole genome sequencing analysis showed that CGMCC 4969 contained nitrile-metabolic enzymes(NHase/amidase and nitrilase)mediated IAA synthetic pathways.The cobalt ion activated the NHase/amidase pathway to accelerate the production of IAA,while the IAN improved the concentration of IAA by activating the nitrilase pathway,when CoCl2 and IAN were respectively added to the culture medium to culture CGMCC4969.CGMCC 4969 NitA had nitrilase activity and IamA had indoleactamide hydratase activity to respectively transform IAN and IAM to IAA,in the two nitrilase(NitA and NitB)and two indoleactamide hydratase(IamA and IamB).Quantitative PCR analysis indicated that CGMCC 4969 NHase/amidase system was constitutively expressed,whereas the nitrilase was inducible.In conclusion,this article reported a novel nutrient concentration induced ACE degradation mechanism by E.adhaerens CGMCC 6315,and its ACE degradation activity was improved with the decreased nutrient concentration.Besides,we also found that the improvement of V.boronicumulans CGMCC 4969 NHase activity required an optimal activator expression level.V.boronicumulans CGMCC 4969 NHase not only participated into the ACE degradation,but also functioned in the IAA synthesis by the coupled amidase and nitrilase.Thus,E.adhaerens CGMCC 6315 and V.boronicumulans CGMCC 4969 have the potential to be applied to ACE remediation in the environment and used as the fertilizer to plant growth.