Isolation And Characteristics Of Pymetrozine-degrading Strains?Analysis Of Its Metabolic Pathways And Cloning Of The Hydrolase Gene

Pymetrozine is a new type of pyridine azomethine insecticide that is widely used to control whiteflies,leafhoppers,and cotton aphids in crops due to its superior insecticidal effect.Because it is widely used worldwide,pymetrozine residues can be detected from water,agricultural products and bee living environments,and thus poses a potential threat to the ecological environment and human health.It has been reported that pymetrozine is highly toxic to aquatic organisms and is genotoxic to human peripheral lymphocytes.The U.S.Environmental Protection Agency(USEPA)has classified pymetrozine as a likely human carcinogen.Therefore,more and more attention has been paid to its environmental behavior and degradation mechanism.Previous studies have demonstrated the hydroxylation of pymetrozine is the major metabolic method in animals.However,bacterial degradation of pymetrozine has not been investigated to date.The main results are as follows:1?Isolation and characterization of the pymetrozine-degrading strainBy enrichment and cultivation,one bacterial strain BYT-1 capable of growing with pyridone as the sole carbon source was isolated from the sample of the wastewater-treating system of a pesticide plant.Strain BYT-1 could completely degraded 2.30 mM pymetrozine in 20 h under the conditions of 30?,pH 7.0,180 rpm and inoculum OD600 nm of 0.2.Its colony on LB plate was smooth and yellowish.Strain BYT-1 was positive for the following activities or reactions:Ala-Phe-Pro arylamidase,L-proline arylamidase,tyrosine arylamidase,and utilization of sucrose,D-mannose,and D-galactose as carbon sources.It was negative for urease,lipase activities,and utilization of D-sorbitol,D-mannitol,and malonate.The 16S rRNA gene sequence of BYT-1 was very similar to those of known Pseudomonas strains,such as P.guariconensis PCAVU11T(99.93%).Based on the above results,BYT-1 was initially identified as Pseudomonas sp..2?Degradation characteristics of strain BYT-1 and analysis of pymetrozine metabolic pathwayStrain BYT-1 was able to degrade pymetrozine at temperatures ranging from 15? to 50?,and the optimal temperature was 30?.It could degrade pyridoxine in the pH range of 5.0-9.0,but the degradation rate of pymetrozine under acidic conditions was lower than that under neutral or alkaline conditions,and the optimum pH value was 7.0.Within a certain range,the degradation rate of pymetrozine was positively correlated with the inoculum size.Strain BYT-1 completely degraded pyridoxine within the concentration range tested(0.46 mM to 2.30 mM).Although the time required was also increased with the increasing concentration,no lag phase was found in the process of degrading high concentration of pymetrozine,indicating that high concentration of pymetrozine had no toxic effect on the strain.Investigation on the degradation pathway by HPLC and MS/MS showed that pymetrozine was initially hydrolyzed to 4-amino-6-methyl-4,5-dihydro-2H-[1,2,4]triazin-3-one(AMDT)and nicotinic acid(NA),the former would accumulate as the end product in the culture,while the latter was hydroxylated to 6-hydroxynicotinic acid(6HNA)and then subjected to further degradation.Strain BYT-1 not only completely degraded 2.30 mM pymetrozine,but also completely degraded 2.30 mM NA and 6HNA,and the strain BYT-1 showed almost the same growth incrementing degradation of the same concentrations of pymetrozine,NA and 6HNA indicating that the carbon source for the growth of the strain BYT-1 actually came from NA,the hydrolysis product of pymetrozine.3?Cloning of the pymetrozine hydrolase gene bytHThe mutant library of BYT-1 was constructed by the method of transposon Tn5 insertion mutagenesis and one mutant named BYT-1M was screened from 12,000 library strains.Strain BYT-1M could use NA as the sole carbon source for growth,but could not use pymetrozine as the sole carbon source for growth,indicating that the gene responsible for the initial degradation step of pymetrozine was disrupted.The upstream and downstream sequences of the Tn5 transposon insertion position in the mutant BYT-1M were amplified by SEFA-PCR,respectively.After sequencing,it was analyzed and spliced to obtain a DNA fragment of 5996 bp in length,and the complete ORF was analyzed separately,and it was found to be a gene cluster containing six genes,which encodes three putative transporters,and three proteins of unknown function(ORF1,ORF2,ORF3),in which the transposon insertion site is located in ORF2,which is 846 bp in length,and amino acid residues encoding proteins is 281 aa in length,30.1 kDa in size.It is speculated that the insertion of Tn5 in ORF2 may cause BYT-1M to lose the ability to degrade pymetrozine,so the gene ORF2 may be involved in the degradation of pymetrozine.The amino acid sequence of ORF2 showed 61.65%similarity to a protein containing the DUF861 domain,and it was named bytH.The bytH gene was cloned into pBBR1MCS-5 to construct the complementary plasmid pBBRl-bytH,which was then introduced into BYT1M by triparental conjugation,and the complemented strain bytH-BYT1M(pBBR1-bytH)was constructed.It restored the ability of degrading pymetrozine,indicating that the gene bytH is responsible for the initial degradation step of pymetrozine.4?Expression and functional identification of pymetrozine hydrolase gene bytHThe bytH gene was ligated to the expression vector pET-29a(+)for heterologous expression in E.coli BL21(DE3)and the recombinant protein was purified by Ni2+-NTA affinity chromatography,and a single band of approximately 30.1 kDa appeared on the SDS-PAGE electropherogram.The enzyme assay of BytH showed that it can break the pymetrozine C=N double bond and convert pymetroin to NA and AMDT.Given that the high similarity to BytH is a protein containing the DUF861 domain,its function is unknown.To study the key amino site of BytH,we selected two conserved amino acid sites for mutation based on the amino acid sequence alignment of BytH,including Trp 121 and Arg 198,and we also selected four non-conservative amino sites for mutations,including Cys 137,Cys 212,Thr 73 and Leu 145.The mutated genes bytHC137A?bytHC212A?bytHW121A?bytHR198A?bytHT73A?bytHL145A?bytHC137A&C212A?bytHT73A&L145A and bytHW121A&R198A were obtained by overlap PCR and constructed into the expression vector pET-29a(+),and then the recombinant plasmid was transformed into E.coli BL21(DE3).After induction,they were tested for their hydrolytic activity against pymetrozine by whole cell catalysis.The results showed that mutations in Trp 121,Cys212 and Cys 137 resulted in the loss of 20.87%,16.32%and 14.66%of hydrolysis activity of BytH,while the mutations of Thr 73,Leu 145 and Arg 198 did not produce impact on the activity of BytH.Simultaneous mutation of Thr 73 and Leu 145 resulted in the loss of 29.84%of pymetrozine hydrolyzing activity of BytH.However,simultaneous mutation of Trp 121 and Arg 198,Cys 137 and Cys212 resulted in complete loss of pymetrozine hydrolyzing activity of BytH.From the above results,it can be concluded that Cys212,Cys 137,Trp 121 and Arg 198 have a great influence on the hydrolysis of pymetrozine by BytH.Further research is needed.