Ensifer Adhaerens CGMCC 6315 Degrades Thiacloprid And Knockdown Of The Transcriptional Regulator NtrC

Thiacloprid(THI)is a widely used neonicotinoid insecticide.However,the extensive application of THI has caused many ecological and environmental problems,such as threaten honey bees,birds and other pollinators and caused pollution to soil and surface water.Microbial degradation is considered to be the most effective and economical method for pesticide remediation in contaminated environments.E.adhaerens CGMCC 6315 is a plant growth-promoting rhizobacteria previously screened by the laboratory,which degrade ACE to amide ACE(IM-1-2),but there is no relevant research on the degradation of THI.In this paper,the metabolic pathway and enzymatic properties of THI degradation by E.adhaerens CGMCC 6315 and the regulation and induction expression of THI metabolic enzyme activity by copper ion were studied,as well as the nitrogen metabolism regulator ntr C of the strain's was knocked out.High performance liquid chromatography(HPLC)and liquid chromatography mass spectrometry(LC-MS)analysis showed that resting cells of E.adhaerens CGMCC 6315 metabolize THI to THI amide,and its metabolic pathway is nitrile hydration.The degradation kinetics of THI by resting cells cultured in LB liquid medium showed that the concentration of THI decreased from 0.59 mmol/L to 0.19mmol/L in 60 h,the degradation rate was 67.80%and the half-life was 36.48 h.The culture conditions were further optimized,the results showed that the resting cells could degrade 85.25%of 0.59 mmol/L THI within 1 h when the LB medium was replaced by MSM medium(adding 2 g/L sodium succinate as carbon source).In MSM medium(2g/L sodium succinate was added as carbon source),2 g/L of different nitrogen sources were added respectively,and the results showed that the addition of ammonium salt and urea significantly inhibited the degradation activity of THI compared with sodium succinate alone.Interestingly,2 g/L of ammonium salt was added to MSM medium(2g/L glucose was added as carbon source)increased the degradation activity of THI.In addition,we also explored the effects of different amino acids on the degradation activity of THI.When 2 g/L of different amino acids were added to MSM medium,the degradation activity of THI was improved.Among them,2 g/L serine was added in MSM medium,the resting cells could decreased THI from 0.59 mmol/L to 0.01 mmol/L within 100 min,the degradation rate reached 98.31%,and the half-life was only 15.40min.The effects of copper on the growth and THI degradation of CGMCC 6315 were determined by adding different concentrations of copper ions into the MSM medium.The results showed that the degradation activity of CGMCC 6315 on THI was increased in different degrees with the increase of copper ion concentration in the culture medium.When the concentration of copper ion increased to 0.5 mmol/L,the degradation activity still increased by 25.68%and the percentage of active strains reached 88.60%(the inhibition rate to the growth of strains was only 11.40%),showing a high copper resistance,and could adsorb 80.10%of 12 mg/L copper within 3 days.When E.adhaerens CGMCC 6315 was inoculated into soil for 12 h,the THI concentration was decreased from 9.38 mg/kg to 1.86 mg/kg,and the degradation rate was 80.17%.After 24 h,the degradation rate reached 96.06%.When E.adhaerens CGMCC 6315 was inoculated into soil with copper content of 12.7 mg/kg for 12 h,the THI concentration was decreased from an initial level of 9.41 mg/kg to 0.17 mg/kg,and the degradation rate was 98.19%.After 24 h,THI was entirely eliminated.The results showed that copper not only had no inhibitory effect on the growth of E.adhaerens CGMCC 6315,but also could increase the degradation rate of THI in copper-contaminated soil.E.adhaerens CGMCC 6315 degraded THI to THI amide by nitrile hydratase(NHase).Enzyme activity analysis showed that the specific activity of Nhp A to THI was 55.77 U/mg,which was 16.4-fold higher than that of Nhc A.The degradation activity of THI by Nhp A was the highest NHase activity reported so far.The V_max of Nhp A toward THI was 90.09?mol/mg/min,which was 14.74-fold higher than that of Nhc A,the K_m were 0.14 mmol/L,indicating that Nhp A had higher affinity for THI than that of Nhc A.The effect of metal ions on the activity of NHase showed that different concentrations of copper(0.03-0.5mmol/L)were added to the enzyme reaction solution,and then the effect of copper on the purified NHase was detected.The results showed that the activity of Nhc A was improved in different degrees.When the copper concentration was 0.05 mmol/L,compared with the control without copper ion,Nhc A activity increased by 2.08 times,while Nhp A did not significantly change,indicating that copper improved THI degradation activity by activating the Nhc A.This differs from many reports that copper had an inhibitory effect on NHase activity.Genomic analysis showed that there were two copper-resistant operons in the E.adhaerens CGMCC6315 genome,which were located on plasmid 1 and plasmid 2,respectively.The expression of copper resistance gene and NHase gene was further determined by q PCR.The results showed that in the presence of 0.1 mmol/L copper,the transcription levels of poc A,poc B and pme R in copper resistance gene cluster 1were increased by 6.35-fold,3.57-fold and 2.24-fold higher than those in the control group without copper,respectively.However,the expression levels of two copper oxidase-encoding genes,poc M and poc N,showed no obvious change.In copper resistance gene cluster 2,the transcriptional levels of poc H,poc R,poc S and poc O were increased by 5.26-fold,2.79-fold,3.21-fold and 2.60-fold respectively.q PCR analysis showed that the transcriptional levels of nhp A was up-regulated by 3.32-fold in the presence of 0.1 mmol/L copper compared with no copper addition,whereas,the expression of nhc A showed no obvious change,indicating that copper improved THI degradation activity was by inducing the expression of nhp A.Previous studies in the laboratory have shown that the synthesis of Nhp A of E.adhaerens CGMCC 6315 is regulated by nitrogen metabolism.Ntr C is a global regulatory factor.The proteome of the two-component regulatory system KEGG pathway related to low nitrogen metabolism showed that the protein numbered6315GL003118 is a?54-dependent Fis family of transcriptional regulators.In order to verify whether the transcriptional regulatory factor 6315GL003118 is involved in the transcriptional regulation of nhp A,the knockout system of wild-type strain E.adhaerens CGMCC 6315 was constructed.The upper and lower arms of6315GL003118 were amplified by overlapping PCR and ligated with suicide vector p EX18Gm.The recombinant plasmid was transferred into E.adhaerens CGMCC 6315by conjugation transfer,and the 6315GL003118 gene defective strain was successfully constructed through two homologous recombination.In conclusion,it is reported for the first time that E.adhaerens CGMCC 6315 is a copper-resistant bacterium with an efficient ability of THI degradation.Copper improved the degradation rate of THI by activating Nhc A and inducing the expression of nhp A,which is a new regulation mechanism of nitrile hydratase that has not been reported at present,and has a good application potential in the treatment of co-contamination environment.The 6315GL003118 gene defective strain was successfully constructed,which laid a foundation for further verification of whether 6315GL003118was involved in the transcriptional regulation of pnhA.