Molecular Mechanism Of Carbaryl Degradation By Pseudomonas Sp.XWY-1

Carbaryl(1-naphthyl-N-methylcarbamate)is a carbamate insecticide widely used in agricultural and forestry pest control.Since carbaryl inhibits the activity of acetylcholinesterase and poses a potential threat to humans and ecology,it has attracted more and more attention.In 2008,the European Chemicals Review Committee listed it as a third type of carcinogen and proposed to ban the use of carbaryl,And China has listed carbaryl as a restricted pesticide,but carbaryl residue could still be detected in surface waters now.Therefore,the carbaryl in the environment needs to be eliminated.Microorganisms play an important role in the degradation of carbaryl.In this study,a carbaryl-degrading strain Pseudomonas sp.XWY-1 was isolated;the degradation pathway of carbaryl in strain XWY-1 was proposed;the carbaryl hydrolase gene was cloned;the key amino acid residues of canbaryl hydrolase were identified;the genome of strain XWY-1 was analyzed;plasmid pXWY was found to harbor a gene cluster related to carbaryl degradation and the horizontal transfer of plasmid pXWY was investigated.The results of this study will help elucidate the molecular mechanism of bacterial degradation of carbaryl.1.Isolation,identification and growth characteristics of a carbaryl-degrading strainA carbaryl-degrading strain XWY-1 was isolated from the carbaryl-contaminated soil by enrichment method.According to its morphological characteristics,physiological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene sequence,strain XWY-1 was preliminarily identified as a member of Pseudomonas sp..The optimal temperature and pH for growth of strain XWY-1 was 30℃ and 7.0,respectively.When the medium volume in the 250 mL-flask was less than 100.0 mL,strain XWY-1 grew well.Strain XWY-1 could utilize glucose,maltose,succinic acid and glycerol as carbon sources for the growth,and glucose was the optimal carbon source.Strain XWY-1 could utilize potassium nitrate,ammonium chloride,urea,peptone,yeast extract and glycine as nitrogen sources for growth,and yeast extract was the optimal nitrogen.2.Degradation characteristics of carbaryl by strain XWY-1 and speculation of the metabolic pathwayStrain XWY-1 could utilize carbaryl as the sole carbon source for growth.At 30℃,strain XWY-1 degraded 50.0 mg·L-1 carbaryl completely within 30 h.The optimal temperature and pH value for carbaryl degradation was 30℃ and pH 7.0,respectively.When the medium volume in the 250 mL-flask was less than 100.0 mL,strain XWY-1 showed good degradation performance against carbaryl.The increase in inoculum promoted carbaryl degradation by strain XWY-1.Only 1-naphthol was detected as the metabolite of carbaryl degradation by strain XWY-1 by HPLC-MS/MS.Therefore,we determined the utilization of key metabolites in the reported typical carbaryl degradation pathway by strain XWY-1,including 1-naphthol,salicylic acid and gentisic acid.The results showed that strain XWY-1 could use these substrates as the sole carbon source for growth.Accordingly,it can be speculated that there is a typical carbaryl degradation pathway in strain XWY-1.Carbaryl is initially hydrolyzed to 1-naphthol,and then converted to salicylic acid,followed by gentisic acid,and finally entered the TCA cycle after a series of steps.3.Cloning and expression of carbaryl hydrolase gene mcbA and identification of key amino acidsThe carbaryl hydrolase is the enzyme responsible for the initial step of the carbaryl degradation pathway.Therefore,we cloned the carbaryl hydrolase gene mcbA from strain XWY-1 by establishing a transposon insertion mutant library,which was 2364 bp in length.The mcbA gene was heterologously expressed in E.coli BL21 and the product was purified.Its size on SDS-PAGE was approximately 80.0 kDa,which was consistent with the predicted size.McbA was identified as a member of esterase family.The optimal conditions for the hydrolysis of carbaryl by McbA were 40℃ and pH 7.0,respectively.Cu2+and Hg2+significantly inhibited the enzyme activity,while Co2+,Ni2+and Zn2+significantly promoted the eRzyme activity,and Ca2+ and Fe2+ promoted the enzyme activity to a certain degree.Mn2+ showed almost no effect on the activity.McbA could hydrolyze a variety of carbamate insecticides,of which the highest activity was aginst isoprocarb,followed by fenobucarb,carbaryl,carbofuran,aldicarb and propoxur.However,the activity of McbA aginst methomyl and oxamyl was not detectable.The apparent Km and kcat values of McbA for carbaryl were 77.67 ±12.31 μM and 2.12±0.10 s-1,respectively.A PHP-like domain was predicted by Interpro between 447aa to 594aa region of McbA.This region contains five histidine residues His451,His464,His467,His477 and His504.The five histidine residues in this region were replaced with glycine residues by overlap PCR,respectively,generating five variants of McbAH451A,McbAH464A,McbAH467A,McbAH477A and McbAH504A.Another variant McbAH467A&H477A was obtained by simultaneously replacing His467 and His477 with glycine residues.And the activity of the variants was determined.McbAH467A,McbAH477A,McbAH504A,McbAH467A&H477A lost 67.32%、21.72%、98.66%and 92.72%of activity,respectively,while the activity of McbAH451A and McbAH464A showed no significant diffence with wild-type McbA.Therefore,His504 is the most critical amino acid residue of McbA,and His467 and His477 also have a great influence on the activity of McbA.4.Genomic analysis of strain XWY-1The genome of strain XWY-1 was sequenced by Shanghai Lingen Biotechnology Co.,Ltd.The genome size of strain XWY-1 was 6,297,253 bp,including one chromosome(5,902,716 bp;G+C%61.9%)and one plasmid pXWY(394,537 bp;G+C%56.3%).Comparative genome analysis indicated a high degree of similarity between the genome of Pseudomonas sp.XWY-1 and the genomes of P.putida KT2440,P.putida F1,P.putida ND6 and Pseudomonas sp.C5pp.The metabolic network of aromatic compounds in strain XWY-1 was analyzed,the result showed that strain XWY-1 can degrade a variety of aromatic compounds,and then the metabolic networks of nitrogen-containing compounds and sulfur-containing compounds in strain XWY-1 were analyzed by KEGG,indicating that strain XWY-1 has metabolic diversity.By comparison with P.putida KT2440,it was found that these two strains showed a high degree of similarity in the metabolic network of these compounds.A gene cluster with 48,876 bp in length was found at 269,196 bp-318,071 bp of plasmid pXWY.It showed high similarity to the reported carbaryl degradation gene cluster mcb(mcbABCDEFGHIJKLMNOPQ)on the chromosome of Pseudomonas sp.C5pp.Therefore,we speculated that plasmid pXWY was responsible for the degradation of carbaryl in strain XWY-1.5.Functional verification and horizontal transfer of plasmid pXWYA plasmid-free mutant strain XWY-NP was obtained by plasmid curing.Strain XWY-NP lost the ability to degrade carbaryl,1-naphthol,salicylic acid and gentisic acid,indicating that plasmid pXWY is responsible for carbaryl degradation in strain XWY-1.The horizontal transfer of plasmid pXWY between strain XWY-1 and the recipient strains Alcaligenes faecalis JQ135,P.putida KT2440,Sphingomonas sp.RW1,Rhodococcus erythropolis PR4,Paenarthrobacter sp.YJN-5 was studied.It was found that pXWY can be transferred to A.faecalis JQ135 and P.putda KT2440,and the resulted strains were named as JQ135-pXWY and KT2440-pXWY,respectively.When the host strain was subcultured in LB medium without addition of carbaryl,pXWY could be stably passed in the original host strain XWY-1,but the stability was not high in JQ135-pXWY and KT2440-pXWY.When passed to the second generation in JQ135-pXWY,the loss rate of plasmid pXWY was above 90.0%,and the plasmid loss rate of KT2440-pXWY was about 60.0%.The addition of carbaryl is beneficial to maintain the stability of plasmid pXWY,and pXWY in KT2440-pXWY was stably passed under 500.0 mg·L-1 carbaryl.Strain KT2440-pXWY can degrade 50.0 mg·L-1 of carbaryl within 30 h,indicating that that pXWY confers its ability to degrade carbaryl This also indicates that pXWY has a certain application potential in the bioremediation of carbaryl polluted environment.