The Catabolism And Its Molecular Mechanism Of 3,6-Dichlorosalicylate In Rhizorhabdus Dicambivorans Ndbn-20

3,6-Dichlorosalicylate(3,6-DCSA)is the demethylation product of herbicide dicamba.Dicamba(3,6-dichloro-2-methoxybenzoic acid)is a low mammalian toxicity,broad-spectrum and high-efficiency hormone herbicide,and widely used to control a variety of broadleaf weeds in corn,sorghum and wheat fields.Especially after the large-scale planting of GM dicamba-resistant crops constructed by Monsanto Biotechnology Company,the annual use of dicamba significantly increased from 15,000 tons to more than 60,000 tons.Thus,the catabolism and ecological effects of dicamba needs to be studied.In the environment,dicamba is mainly degraded through microbial metabolism.In reported dicamba-utilizing bacteria,dicamba is initially demethylated to generate 3,6-dichlorosalicylate(3,6-DCSA).3,6-DCSA is a toxic and persistent chlorinated aromatic compound that has potential hazards to the ecological environment and human health as it accumulates in soil and agricultural products.However,the catabolic pathway and the genes and enzymes involved have remained unknown.Therefore,it is of great theoretical and practical value to elucidate the microbial catabolic pathway of 3,6-DCSA and the enzymes and genes involved.In this study,a dicamba-degrading' strain Rhizorhabdus dicambivorans Ndbn-20 was used as the research material,we revealed the microbial catabolism of 3,6-DCSA and the genes and enzymes involved.The main results obtained are as follows:1.Screen of the 3,6-DCSA-degradation-deficient mutant and the preliminary identification of the degrading gene clustersA 3,6-DCSA-degradation-deficient mutant,Ndbn-20m,was acquired by continuous transfers of the strain on 1/5 Luria-Bertani(LB)agar without addition of 3,6-DCSA.Pairwise comparison of the mutant genome with that of the wild-type strain revealed that a 64.1-kb DNA fragment was absent in mutant Ndbn-20m.ORF analysis showed that there was a gene cluster,designated dsmR1DABCEFGR2(3,6-DCSA monooxygenase),existed in the 64.1-kbfragment.dsmABC encoded a three-component cytochrome P450,dsmD,dsmE,dsmF and dsmG encoded gentisate dioxygenase,fumarylpyruvate hydrolase,transporter and maleylacetate reductase,respectively.Genes dsmR1 and dsmR2 encoded LuxR family transcriptional regulator and IclR family transcriptional regulator,respectively.RT-PCR results showed that dsmD,dsmA,dsmB,dsmC,dsmE,dsmF,dsmG and dsmR1 were organized in an operon.RT-qPCR results showed that all the genes in cluster dsm were significantly induced by 3,6-DCSA.In addition,we found that another gene cluster,which contained a gentisate dioxygenase gene gtdA and a glutathione-dependent dehalogenase gene dsmH2,was also obviously induced by 3,6-DCSA.2.The three-component cytochrome P450 monooxygenase DsmABC was responsible for the 5-hydroxylation of 3,6-DCSADsmA shared 30%-36%identities with some cytochrome P450 monooxygenases that catalyze the hydroxylation of antibiotics.In the phylogenetic tree constructed by neighbor-joining(NJ)algorithm based on related cytochrome P450 monooxygenases,DsmA was located within this NJ tree,but formed a monophyletic branch.DsmB and DsmC were ferredoxin and ferredoxin reductase,respectively,and constituted an electron transfer chain.Fragments containing dsmA or dsmABC were ligated into the broad-host-range plasmid pBBR1MCS-2,and then introduced into Sphingobium quisquiliarum DC-2,which could not degrade 3,6-DCSA and salicylate.Recombinant DC-2(pBBR-dsmABC)acquired the ability to transform 3,6-DCSA,and correspondingly,a hydroxylation product was produced.Recombinant DC-2(pBBR-dsmA)could not transform 3,6-DCSA.Mutant Ndbn-20?dsmA could not degrade 3,6-DCSA.Re-introduction of dsmA into Ndbn-20?dsmA restored the.ability to degrade 3,6-DCSA.Single-crystal X-ray diffraction analysis showed that the DsmABC-catalyzed hydroxylation occurred at the C-5 of 3,6-DCSA,forming 3,6-dichlorogentisate(3,6-DCGA).The above results indicated that DsmABC was a 3,6-DCSA 5-hydroxylase.3.The glutathione-depende-nt dehalogenase DsmH2 was responsible for the 6-dechlorination of 3,6-DCGAThe cell lysate of strain Ndbn-20 could transform 3,6-DCGA only in the presence of GSH,indicating that 3,6-DCGA was dechlorinated by a glutathione S-transferase(GST)in strain Ndbn-20.By genome comparison and blast,two GSTs,dsmH1and dsmH2,were found in the genome of strain Ndbn-20.Enzymatic results showed that both DsmHland DsmH2 displayed 3,6-DCGA dehalogenase activity.DsmH2 had significantly higher catalytic efficiency toward 3,6-DCGA than DsmH1.Transcription and disruption analysis revealed that DsmH2,but not DsmHl,was responsible for the 6-dechlorination of 3,6-DCGA in strain Ndbn-20 in vivo.1H NMR analysis demonstrated that the 6-chlorine but not the 3-chlorine of 3,6-DCGA was replaced by a hydrogen,generating 3-chlorogentisate.Furthermore,we propose a novel 'eta' class of GSTs to accommodate the four bacterial dehalogenases PcpC,LinD,DsmH1,and DsmH2.4.The gentisate 1,2-dioxygenase DsmD was mainly responsible the cleavage of 3-chlorogentisateThere were two gentisate 1,2-dioxygenase genes,dsmD and gtdA,existed in the genome of strain Ndbn-20,Enzymatic study showed that both DsmD and GtdA could catalyzed the cleavage of gentisate and 3-chlorogentisate.DsmD had significantly higher catalytic efficiency toward 3-chlorogentisate than gentisate,and than that of GtdA;and GtdA had significantly higher catalytic efficiency toward gentisate than 3-chlorogentisate.These results indicated that the optimal substrate for DsmD is 3-chlorogentisate,while the optimal substrate for GtdA is gentisate.Transcription and disruption analysis revealed that DsmD,but not GtdA,was mainly responsible for the cleavage of 3-chlorogentisate,and GtdA was mainly responsible for the cleavage of gentisate in strain Ndbn-20 in vivo.5.The reductive dehalogenase DsmG catalyzed the reductive dechlorination of 2-chloromaleylpyruvatedsmG shared the highest identity with maleylacetate reductase TfdF(39%).TfdF could catalyze the reductive dechlorination of 2-chloromaleylacetate,and 2-chloromaleylpyruvate,the cleavage product of 3-chlorogentisate,was structurally similar to 2-chloromaleylacetate.Enzymatic study showed that the exogenously expressed and purified DsmG could catalyzed the reductive dechlorination of 2-chloromaleylpyruvate to maleylpyruvate in the presence of NADH.Base on the above results,we basically elucidated the catabolic pathway of 3,6-DCSAin strain Ndbn-20.Two gene clusters are involved in this pathway.3,6-DCSA is 5-hydroxylated to 3,6-DCGA by monooxygenase DsmABC,3,6-DCGA is subsequently 6-dechlorinated to 3-chlorogentisate by dehalogenase DsmH2,3-chlorogentisate is further cleaved to 2-chloromaleylpyruvate by gentisate 1,2-dioxygenase DsmD.2-chloromaleylpyruvate was reductively dechlorinated to maleylpyruvate by a reductive dehalogenase DsmG.Further study is needed to elucidate the catabolism of maleylpyruvate in strain Ndbn-20.