The Cloning And Functional Identification Of Genes PdmAB And DdhA Involved In The Bacterial Degradation Of Phenylurea Herbicides

The phenylurea herbicides,first discovered and marketed in the mid-20th century,have become one of most important and extensively used classes of herbicides in different regions of the world in a short time.The use of this herbicide is increasing,resulting in varying degrees of environment pollution.Ecotoxicological data have suggested that phenylurea herbicides as well as some of their degradation metabolites are harmful not only for animals,plants and microbial activities but also for humans;thus,the fate of phenylurea herbicides in the environment has received considerable attention.Microbial activities play a primary role in the degradation of phenylurea herbicides.Considering the harmful effects of phenylurea herbicides,great concerns have been raised about the degradation pathway and degradation mechanism of phenylurea herbicides.Phenylurea herbicides harbor two kinds of substitutes branched on the phenyl ring;either N,N-dimethyl substitutes or N-methoxy-N-methyl-substituted compounds.Up to now,several microbial strains have been isolated and characterized for phenylurea herbicides degradation.Notably,some genera seemed to have an extraordinary ability to degrade certain phenylurea herbicides.For instance,despite different geographical origins,most N-methoxy-N-methyl-substituted phenylurea herbicide catabolizing isolates belong to the genus Variovorax,while most of the strains reported to be able to mineralize N,N-dimethyl-substituted phenylurea herbicides are Sphingomonads(bacteria of the genus Sphingomonas and the closely related genera Novosphingobium,Sphingopyxis,and Sphingobium are commonly referred to as sphingomonads).There are many research reported which indicated the degradation pathways as well as the molecular mechanism on the degradation of N-methoxy-N-methyl-substituted phenylurea herbicide by strains of Variovorax,and the N,N-dimethyl-substituted phenylurea herbicide degradation pathways of sphingomonads was clear.However,the degrading-related genes of this pathway have not yet been characterized.In our previous study,Sphingobium sp.YBL2 was isolated as a degrader of IPU and other phenylurea herbicides.The typical mineralized pathway of N,N-dimethyl-substituted phenylurea herbicide in bacterial strain YBL2 includes two successive N-demethylations,cleavage of the urea side chain and aromatic ring,and ultimately mineralized to CO2 and production of biomass.In this paper,we took strains YBL2 and IPU on as the object of the research,aimed to investigate the degradation mechanism of N,N-dimethyl-substituted phenylurea herbicide.This article will launch the research from following several aspects.1.Cloning and functional analyse of the gene pdmAB responsible for N-demethylation in strain YBL2Sphingobium sp.strain YBL2,isolated from China,were found to be able to mineralize commonly used N,N-dimethyl-substituted phenylurea herbicides.Generally,the microbial metabolism of N,N-dimethyl-substituted phenylurea herbicides is initiated by a mono-N-demethylation event.In addition,the initial N-demethylation was found to be the rate-limiting step for mineralization of IPU.In this study,the molecular basis for this process was revealed.The pdmAB genes in Sphingobium sp.strain YBL2 were shown to be responsible for the initial mono-N-demethylation of commonly used N,N-dimethyl-substituted phenylurea herbicides.PdmAB is the oxygenase component of a bacterial Rieske non-heme iron oxygenase(RO)system.The genes pdmAB,encoding the a subunit PdmA and the ? subunit PdmB,are organized in a transposable element flanked by two direct repeats of an insertion element resembling ISRh1.Furthermore,this transposable element is highly conserved among phenylurea herbicide-degrading sphingomonads originating from different areas of the world.However,there was no evidence of a gene for an electron carrier(a ferredoxin or a reductase)located in the immediate vicinity of pdmAB.Without its cognate electron transport components,expression of PdmAB in Escherichia coli,Pseudomonas putida,and other sphingomonads resulted in a functional enzyme.Moreover,coexpression of two putative[3Fe-4S]-type ferredoxin from Sphingomonas sp.strain RW1 or Sphingobium sp.strain YBL2 greatly enhanced the catalytic activity of PdmAB in E.coli.These data suggested that PdmAB has a low specificity for electron transport components and that its optimal ferredoxin may be the[3Fe-4S]type.PdmA exhibited low homology to the a subunits of previously characterized ROs(less than 37%identity)and did not cluster with the RO group involved in O-or N-demethylation reactions,indicating that PdmAB is a distinct bacterial RO N-demethylase.2.Cloning and expressing the hydrolase gene ddhA and the study on the enzymatic properties of ddhAThe DDIPU-hydrolase gene,designated ddhA,were cloned from Sphingobium sp.strain YBL2 by comparing the draft genome of YBL2 with its mutant YBL2-Mut which could not degrade DDIPU,indicating that the gene responsible for cleavage of the urea side chain of phenylurea herbicides was deleted or disrupted.Sequence analysis indicated that ddhA consists of 2139 bp,encoding a protein of 712 amino acids,the G+C mol%content was 47.5%.In the NCBI nucleotide databases,there were no similar DNA sequences as the ddhA.The deduced protein showed low sequence identity with the carbaryl-hydrolase CehA from Rhizobium sp.AC 100(14%).DdhA was successfully expressed in Escherichia coli C43(DE3)and purified using Ni-nitrilotriacetic acid affinity chromatography.The molecular mass of DdhA was approximately 80 kDa.One metabolite appeared during DDIPU degradation by the enzyme DdhA and was identified as 4IA on the basis of HPLC and MS/MS analyses.The recombinant protein exhibited optimal activity around 35? at 7.0.Hg2+?Cu2+ and Zn2+significantly inhibited the enzyme activity at 1 mM.In this work,we also studied the substrate spectrum of DdhA.Results showed that the DdhA can degrade MDIPU and 4-chlorophenylurea to generate 4IA and 4-chloroaniline,respectively.DdhA can degrade MDIPU to generate 4IA directly which suggested that the intermediate metabolite MDIPU has two metabolic pathway in YBL2:one was further N-demethylation to generate DDIPU;Another metabolic pathway resulted in the direct accumulation of 4-IA based on the cleavage of the methylurea group of MDIPU.It was shown that DdhA is primarily responsible for leavage of the urea side chain of MDIPU and DDIPU in the process of degrading isoproturon by the strain YBL2,and isoproturon could be transformed to aniline derivatives acted together by PdmAB and DdhA.The structure of 4-chlorophenylurea was similar to DDIPU,and it was the intermediate metabolite of monuron,DdhA was able to degrade 4-chlorophenylurea and the gene is highly conserved in several isoproturon-degrading strains,indicated that DdhA may be the key gene in the typical pathways of microbial degrading N,N-dimethyl substituted urea herbicides,and it had the important theoretical significance to degrade such herbicides.Besides,we found DdhA is able to degrade carbaryl and carbofuran to generate 1-naphthol and 2,3-dihydro-2,2-dimethyl-7-benzofuranol,respectively.3.Isolation and identification of a phenylurea herbicides-degrading bacterium and the degradation pathway of isoproturon and linuron in strain Pu21In this study,a phenylurea herbicides-degrading bacterial strain,named Pu21,were isolated from herbicides manufacturing company by the conventional enrichment culture technique.Strain Pu21 was able to completely metabolize 20 mg L"1 of isoproturon and linuron within 7 days.Based on the phenotypic,physiological,and biochemical tests and phylogenetic analysis,strain Pu21 was characterized as Sphingobium.sp.We studied the degradation pathway of isoproturon and linuron by strain Pu21.The results showed that isoproturon was degaraded by mono-N-demethylation to yield MDIPU,MDIPU was transformed to DDIPU and then cleavage of the urea side chain to generate 4IA,the methylurea group of MDIPU could also be cleave to accumulate 4IA directly.One linuron degradation intermediate metabolite 3,4-DCA was detected during the HPLC and MS/MS analysis.Based on the results above,we proposed a catabolic pathway of linuron which starts with amide hydrolysis to 3,4-DCA,then 3,4-DCA was completely degraded by the strain.