Isolation&Characterization Of A 3,5,6-trichloro-2-Pyridinol(Tcp)-Degrading Bacterium And Cloning&Expression Of Genes Responsible For Its Dechlorination

With the increasing restrictions or eliminations of highly toxic organophosphorus compounds,chlorpyrifos is widely used in agricultural fields in the recent years.3,5,6-Trichloro-2-pyridinol(TCP)is the primary metabolite of both chlorpyrifos and chlorpyrifos-methyl.Besides,TCP is also present in the raw,untreated wastewater of chlorpyrifos-manufacturing or chlorpyrifos-methyl-manufacturing plants.TCP is a charged molecule at neutral pH,so it is more mobile in soil and more leachable into groundwater and surface water,which causes widespread contamination in soils and aquatic environment.Although TCP exhibits low to moderate toxicity to aquatic and terrestrial biota,it had relatively high antimicrobial effects on the microorganisms,which not only prevents its own degradation by microorganisms but also limits the degradation of its parent compound.Concentrations higher than 50 mg/L in the soil have been reported to inhibited microbial degradation of other organic chemicals.Bioremediation has received increasing attention as a reliable,efficient,safe,cost-effective and promising biotechnological approach to cleaning up polluted environments.Although mineralization of TCP has been studied in soil,only a few strains were reported to have the ability to degrade TCP.Most of bacterial strains capable of degrading TCP only degraded low concentration of TCP(50-100 mg/L)within the range of 4 days to 3 weeks.However,only a few reports focus on the degradation metabolites of TCP.In addition,little information is available on the genetic and enzymatic aspects involved in TCP degradation.The main objective of this study is to isolate highly effective TCP-degrading strains,to clone genes responsible for TCP degradation,to express heterologously genes in Escherichia coli and to investigate the biological characteristics and degradation mechanisms of enzymes against TCP.1.Isolation and identification of a TCP-degrading bacteriumStrain P2 was isolated by using the enrichment method from a wastewater treatment system of a pesticide manufacturing company.It could use TCP as the sole carbon source and energy source for its growth.It showed a high average degradation rate.Strain P2 was able to completely metabolize 100 mg/L of TCP within 10 h and completely degrade 800 mg/L of TCP in 70 h,which might be one of the most effective TCP degraders to date.Its 16S rRNA gene sequence showed high similarity with the strain from the genus of Cupriavidus,and the highest similarity of 99.7%was with strain Cupriavidus pauculus LMG 3413.Based on the phylogenetic analysis of the 16S rRNA gene and the physiologic&biochemical characteristics,strain P2 was identified as Cupriavidus sp.and named as Cupriavidus sp.P2.This is the first report of TCP-degrading strain from the genus of Cupriavidus,which enriches the diversity of TCP degraders.2.Study on the growth and degradation characteristics of the TCP-degrading bacteriumThe optimal temperature and initial pH of medium for growth of strain P2 were 30 ?and pH 7.0,respectively.The optimal carbon source and nitrogen source were glucose and peptone,respectively.The optimal concentration of NaCl for growth of strain P2 was 5 g/L.The growth of strain P2 was positively correlated to the ventilation quantity.Strain P2 can endure 100 g/L of ampicillin.Strain P2 was able to mineralize both TCP and 2,4,6-trichlorophenol.During the degradation of TCP,two colorful metabolites(Rt=3.737 min and 8.809 min,respectively)were detected.The light-green phenomenon was also reported in Ralstonia sp.T6,but the green-yellow phenomenon was found for the first time.We tried our best to analyze the metabolites by mass spectrum,but all failed,so further investigation is needed to identify the accurate structure of the metabolites.The optimal temperature and pH for TCP degradation were 30 ? and pH 8.0,and the optimal concentration of NaCl was 5 g/L.The degradation efficiency increased positively with the increase of ventilation quantity and inoculation amount.These results are consistent with the corresponding conditions of optimal growth of strain P2.The addition of metal ions(1 mmol/L)had different effects on TCP degradation when strain P2 grew in mineral salts medium supplemented with 100 mg/L TCP.Co2+,Zn2+and Ni2+ seriously inhibited the degradation of TCP.Fe2+,Mn2+and Ca2+ had no significant influence on the degradation of TCP.Surprisingly,Cu2+ completely inhibited the degradation of TCP.3.Cloning and analyse of the gene cluster responsible for TCP dechlorinationThe Tn5-derived mini-transposon of a plasmid vector pSC123 was successfully introduced into.the chromosome of strain P2 via electroporation.Three positive clones(designated as strains P2-M1,P2-M2,and P2-M3)were screened from approximately 7800 transformants.HPLC detection showed that they lost the ability to degrade TCP.The genomic DNA of the three mutants was used as the template for SEFA-PCR to clone genes that flank the kanamycin resistance gene.In addition,the products were cloned to pMD18-T and subjected to DNA sequencing.The results show that the DNA fragment upstream of kanamycin resistance gene in strains P2-M1,P2-M2,and P2-M3 were 1538 bp,2165 bp,and 3021 bp,respectively;The DNA fragment downstream of kanamycin resistance gene were 2862 bp,2216 bp,and 2109 bp,respectively.Finally,a continuous 6012 bp gene cluster was obtained based on the splicing of all these fragments.The sequence was analyzed using online ORF Finder and BLASTx program(www.ncbi.nlm.nih.gov).The orfl gene(complement 5-841)encodes FMN adenylyltransferase,which showed the highest sequence identity with FMN adenylyltransferase from C.necator N-1(85.92%)and Ralstonia eutropha JMP134(83.33%).tcpRl(complement 933-1904)encodes LysR-type transcriptional regulator,which showed the highest sequence identity with the LysR-type transcriptional regulator protein of Ralstonia sp.T6(99.38%)and Ralstonia eutropha JMP134(89.78%).tcpBl(2053-2604)encodes flavin reductase,which showed the highest sequence identity with the flavin reductase from Ralstonia sp.T6(98.91%),Cupriavidus necator N-1(88.46%)and Ralstonia eutropha JMP134(88.11%).tcpAl(2709-4262)encodes a monooxygenase,which has the highest sequence similarity with the monooxygenase from Ralstonia sp.T6(99.37%),C.necator N-1(96.64%),and Ralstonia eutropha JMP134(96.64%).orf2(4633-5502)encodes maleylacetate reductase,which showed the highest sequence identity with the maleylacetate reductase from Ralstonia sp.T6(99.65%)and C.necator N-1(92.01%).Analysis of the inserted site of kanamycin resistance gene in P2-M1,P2-M2,and P2-M3 revealed that they were 1326 bp to1327bp locus(located in the interior site of tcpRl gene),2139 bp to 2140 bp(located in the interior site of tcpBl gene),and 4153 bp to 4154 bp(located the interior site of tcpA1 gene),respectively.All of them lost the ability to degrade TCP.Therefore,the tcpA1,tcpBl,and tcpRl genes are related to the degradation of TCP.This is the first report of a gene cluster responsible for TCP degradation.4.Study on the expression and the product characterization of genes responsible for TCP dechlorinationThe genes tcpAl and tcpBl of strain P2 were cloned from genomic DNA of strain P2 by PCR.The PCR products were ligated into pET-29a to generate the recombinant plasmids(pET-tcpA1 and pET-tcpBl)and then transformed into E.coli BL21.However,the transformants with pET-tcpA1 or pET-tcpBl could not degrade TCP,even when FAD and NADH were added into the medium.But the mixture of transformant with pET-tcpA1 and transformant with pET-tcpBl completely degraded 0.5 mM TCP within 10 h,which suggested that TcpAl and TcpBl were together responsible for the degradation of TCP in strain P2.The genes of tcpBl and tcpAl were expressed in E.coli BL21,and the two enzymes with His-tag tails(TcpA1 and TcpB1)were purified using Ni-nitrilotriacetic acid affinity chromatography,respectively.However,purified TcpA1 alone or TcpB1 alone was unable to degrade TCP.TCP was only degraded when they worked together,indicating that TcpAl and TcpBl were indispensable for the dehalogenation of TCP in strain P2.It was the first time to elucidate that TcpAl and TcpB1 worked together to catalyze the dehalogenation of TCP.The purified enzymes were analyzed via SDS-PAGE and appeared as a single band.The denatured TcpB1 was 19.0 kDa,whereas TcpAl was 58.5 kDa,which are consistent with the corresponding predicted molecular mass.Therefore,TcpAl and TcpBl were purified well and regarded as pure enzymes for further study.The substrate specificity of TcpAl for TCP and 2,4,6-trichlorophenol was investigated.TcpAl was able to transform both substrates.TcpAl has a lower Km(34.75 ?M)and a higher catalytic efficiency(kcat/Km)(1.01 ?M-1s-1)for TCP than for 2,4,6-trichlorophenol,which suggested that TCP was a good substrate for TcpA1.Enzyme activity against TCP and 2,4,6-trichlorophenol were investigated in 1 mL of 50 mM PBS buffer(pH 7.5)containing 20?M FAD,10 mM NADH,and 1 mM substrates at 30 ? for 20 min.The reactions were initiated by the addition of purified TcpAl and TcpB1 to a final concentration of 0.02 mg/mL,and terminated by adding 0.02 mL of 30%HNO3.The effects of temperature,pH,and metal ions on TcpAl activity were also investigated.TcpAl showed maximal activity at 30 ?.TcpAl retained low activity under pH 5.0 and the highest activity appeared in pH 8.0.The enzyme showed relatively high activity at pH ranging from 7.0 to 9.0.Cu2+,Zn2+,and Hg2+ significantly inhibited the enzyme activity at 0.2 mM and at 2 mM.Ca2+,Mn2+,and Mg2+ did not significantly influence enzyme activity.Approximately 30%to 60%of TcpAl activity was inhibited by Ni2+ and Co2+ at the tested concentrations.Fe2+ slightly activated the enzyme at 0.2 mM,but slightly inhibited enzyme activity at 2 mM.Adding TcpBl and TcpAl into the reaction mixture containing TCP(0.5 mM)produced a green metabolite.Once TCP was completely transformed,the molarity of the total released chloride ions(1.5 mM)was three times that of the initial TCP in the reaction buffer.Thus,the green metabolite could have originated from the removal of three chlorine atoms from TCP and the product was identified as 3,6-dihydroxypyridine-2,5-dione.TcpAl and TcpBl were found for the first time to work together to convert TCP into 3,6-dihydroxypyridine-2,5-dione.The study of TcpAl characteristics was to elucidate possible mechanisms of TCP degradation of microbiology,and to support theoretical basis for soil bioremediation.5.Preliminary study on bioremediation of TCP-contaminated soil by strain P2The bioremediation of TCP-contaminated soil by inoculating strain P2 was preliminarily studied under laboratory condition.The results showed that strain P2 could promote markedly the degradation of TCP in soil.The optimal degradation condition of TCP in soil was:inoculation cells of strain at 1×106 CFU/g dry soil,pH 7.0,30? and 30%of water content.Under this condition,strain P2 could completely degrade 100 mg/kg TCP in the soil within 15 d.Moreover,the degradation of TCP in soil was promoted due to the growth of fragrant-flowered garlicn in TCP-contaminated soil.Therefore,strain P2 might be a promising candidate for its application in the bioremediation of TCP-contaminated environments.