Identification Of A Novel Thiobencarb Metabolic Pathway In Isolated Bacterium Acidovorax Sp. T1 And Cloning Of Key Genes Responsible For Thiobencarb Biodegradation

Thiobencarb(S-(4-chlorobenzyl)N,N-diethylthiocarbamate),a broad-spectrum,highly efficient and selective thiocarbamate herbicide,is widely used to control grass weeds and some broadleaf weeds,especially barnyard grass and needle spikerush in rice paddies.Thiobencarb is highly toxic to aquatic organisms,amphibians and invertebrates,and its residue in environment also leads to negative impacts on the community structures of algaes and microbes.Thiobencarb have been traced in air,soil,rivers and organisms.Microbial metabolism supplies a crucial approach for thiobencarb degradation in the environment.However,there are limited reports on thiobencarb-degrading microbes,and neither thiobencarb metabolic pathways by strain in pure culture level nor related degrading enzymes and genes have been reported.Therefore,screening for highly effective thiobencarb-degrading strains and exploring corresponding degradation mechanism from cell,gene and enzyme levels would be significant to clarify thiobencarb environmental behaviors.In this study,a thiobencarb-degrading bacterium,designated T1,was isolated from activated sludge by microbial enrichment technology.The degradation characteristics of strain T1 were studied,and two thiobencarb-degrading intermediates were identified by HPLC and GC-MS.A mutant of strain T1,named T1m which lost ability to degrading thiobencarb,was isolated during culture process.A novel two-component FMN-dependent thiobencarb monooxygense gene tmoAB,a NAD+ dependent 4-chiorobenzaldehyde dehydrogenase gene tmoC and a gene cluster encoding a series of genes responsible for 4-chlorobenzoic acid degradation were cloned by comparing and analyzing the genomes of strain T1 and T1m.tmoA,tmoB and tmoC were successfully expressed in E.coli BL21(DE3)respectively,and the characteristics of these three purified fusion proteins were identified.The role of 4-chlorobenzoic acid degradation gene cluster in thiobencarb degradation of strain T1 was revealed from the level of gene transcription,it was the first time that a complete thiobencarb degradation pathway was clarified at biochemical and genetic levels.The main results were as following:1.Isolation and identification of a thiobencarb-degrading bacterium and its degradation characteristicsA thiobencarb-degrading bacterium strain T1 was isolated from activated sludge and was preliminarily identified as Acidovorax sp.T1 by morphological,physiological and biochemical characteristics and 16S rRNA gene phylogenetic analysis.Strain T1 produced clear degradation transparent circles when grown on 1/5 LB agar plate containing thiobencarb.Strain T1 could degrade up to 95.4%of 0.4 mM thiobencarb within 36 h in mineral salts medium(MSM),and grown with thiobencarb as the sole carbon,nitrogen and sulfur source.A mutant strain of T1,named T1m which lost the ability to degrade thiobencarb,was screened by continuous subculture of strain T1 without thiobencarb as a selective pressure.4-chlorobenzaldehyde and 4-chlorobenzoic acid were identified as two thiobencarb degradation metabolites of strain T1 by HPLC and GC-MS.The optimal temperature and pH for the thiobencarb degradation of strain T1 was 30? and 7.0.Strain T1 had high substrate specificity and no degradation activity against other thiocarbamate herbicides such as molinate,vernolate and asulam.Despite of the disability on thiobencarb degradation,strain T1m could still degrade 4-chlorobenzoic acid,4-hydroxybenzoic acid and protocatechuic acid.A bacterium strain of genus Phenylobacterium,named BUT-10,was also isolated during the screening.Strain BUT-10 shared the highest 16S rRNA gene sequence similarity of 97.49%with Phenylobacterium muchangponense A8T,followed by 97.14%similarity with Phenylobacterium immobile DSM 1986T.It was eventually identified as a new species of genus Phenylobacterium by multiphase classification,and the type strain was Phenylobacterium kunshanense BUT-10T(=CCTCC AB 2013085T=KCTC 42014T).2.Gene cloning and function identification of Thiobencarb monooxygense TmoABThe complete genome of strain T1 and the draft genome of strain T1m were sequenced,it was confirmed that an 8988 bp gene fragment on plasmid P3 of strain T1 lost in mutant strain T1m by genomic comparison analysis and PCR identification.A novel two-component FMN-dependent thiobencarb monooxygense gene system tmoAB was cloned from the missing gene fragment.The oxidase component tmoA encoded a protein containing 518 amino acids,and shared the highest amino acid sequence similarity of 32%with Pyrimidine monooxygenase RutA(P58759.1).Combined with the phylogenetic analysis results,TmoA was identified as a new number of group C flavin dependent monooxygese.The reductase component tmoB,located upstream 523 bp of tmoA,encoded 186 amino acids.The phylogenetic analysis revealed that TmoB was homologous to a series of NADH-Flavin reductases with the amino acid sequence similarity of 25%-37%,and alone formed a sub-branch in phylogenetic tree,thus it was identified as a novel flavin reductase.Gene fragment tmoA,tmoB or tmoA-B was ligated into the host vector pBBR1MCS-2,respectively,and the recombinant expression vectors were further introduced into both E.coli DH5a and mutant strain T1m.Whole-cell transformation experiments indicated that the recombinant strains containing pBBR-tmoA or pBBR-tmoA-B restored thiobencarb-degrading ability and could grow using it as the sole carbon,nitrogen and sulfur source.Oxidase component tmoA alone transformed into E.coli DH5a or T1m still exhibited thiobencarb-degrading activity revealing that TmoA has a lower specificity for reductase component.Real-time qPCR analysis showed that the transcription of tmoA was slightly induced by thiobencarb,whereas tmoB were not suggesting that the expression type of tmoA and tmoB were induced and constitutive,respectively.3.Characteristics of purified TmoABThe enzymatic assays showed that TmoB was a NADH-dependent flavin reductase,TmoA was FMNH2-dependent monooxygenase,and the results of functional tests in vitro showed that only the mixture of TmoA and TmoB could convert thiobencarb,suggesting that TmoA and TmoB was a two-component monooxygenase system.TmoA catalyzed the cleavage of the thioester bond of thiobencarb to generate 4CDA and diethylcarbamothioic S-acid,with FMNH2 as electron donor provided by reductase component TmoB.The optimal molar ratio of TmoA and TmoB was 20:1,and the optimal reaction temperature and pH was 20? and 7.4.TmoAB activity was significantly promoted by catalase,and severely inhibited by 1 mM Hg2+,Ni2+,Cd2+,Zn2+and Cu2+and moderately inhibited by 1 mM Ba2+,Mn2+,and Co2+,as well as 5 mM DTT or EDTA.TmoAB showed a high specificity for thiobencarb,and no catalytic activity to other thiocarbamate herbicides and other N or S compounds.4.Gene cloning and characterization of 4-chlorobenzaldehyde dehydrogenase TmoCA NAD+-dependent 4-chlorobenzaldehyde dehydrogenase gene tmoC,located 7129 bp downstream of tmoAB was cloned by comparative genomics and ORF analysis,TmoC consisted of 515 amino acid residues,and shared the highest amino acid sequence similarities 48%-42%with benzaldehyde dehydrogenase XylC(P43503.1)and 4-hydroxybenzaldehyde dehydrogenase PHBDD(P59702.2),and clustered with XylC and PHBDD in the phylogenetic tree.The transcription level of tmoC in cells induced by thiobencarb or 4CDA increased 7.8-and 5.5-fold,respectively,compared to unindued cells.tmoC was highly expressed in E.coli BL21(DE3)by using expression system pET24b(+)and purified by Co2+chelating column to obtain the fusion protein TmoC.The enzymatic assays showed that purified TmoC could dehydrogenated 4CDA to 4CBA using NAD+as a cofactor.5.Functional identification of 4-chlorobenzoic acid degradation gene clusterA complete 4-chlorobenzoic acid degradation gene cluster was cloned from the chromosome of strains T1 and T1m,comprising three operons linked by several transposase genes,and they were responsible for 4-chlorobenzoic acid dechlorination(fcbB-A-T1-T2-T3-C),4-hydroxybenzoic acid hydroxylation(pobA-R)and protocatechuic acid 4,5-cleavage degradation(ligAB-C-I-J-K),respectively.Real-time qPCR analysis showed that the transcription of the key genes fcbB,pobA,ligA and ligB in the three operons were induced by thiobencarb and 4CDA,revealing that the gene cluster was involved in the degradation process of thiobencarb.