| Linuron is one of the most important phenylurea herbicides(PHs)and is mainly used for prevention and control of broad-leaved weeds in food crops.Since its invention,it has become one of the most important herbicides and widely used all over the world.Linuron and its metabolites can disrupt endocrine system,and also have risks of cancer.Meanwhile,it has toxicity for a variety of aquatic organisms and soil organisms.The residue period of linuron in the soil can be 10 days to several years.In recent years,due to its widely use and relatively high water-solubility,linuron and its metabolites have been detected in many sites of the world,including surface water,groundwater,and even in drinking water.The European Union,the United States and other countries and regions have listed linuron as one of the priority control pollutants.Microbial degradation is considered as the major route of the dissipation of linuron in environments.At present,researches about microbial degradation of linuron were mainly focused on the initial degradation of linuron by pure culture strains.No amidohydrolase genes with broad spectrum(can degrade both of N-methoxy-N-methyl and N,N-dimethyl phenylurea herbicides)have been cloned from gram-negative bacteria.The community structure and function of the linuron mineralization culture has not been elucidated.Therefore,strengthening the study of microbial metabolic mechanism and key community structure involved in linuron mineralization has an important scientific significance and application potential.The purpose of this study is to illuminate the community structure and function of the linuron degrading consortium by the means of molecular ecology,and isolate pure strains from the microbial consortium for each step of linuron degradation,reveal the degrading genes,enzymes,metabolic mechanism by the means of physiology and molecular biology.This study will provide strains,gene resources and technical support for the bioremediation of environments contaminated with linuron.1.Acquisition of linuron-degrading consortium and the isolation of degrading strainsA linuron degrading microbial consortium was obtained from an activated sludge collected from a PH producing factory in Jiangsu by the conventional enrichment culture technique.It was found that diuron was completely degraded by the microbial consortium.High-throughput sequencing technology analysis of the community composition of the microbial consortium revealed that Methylobacillus was the predominant genus of the microbial consortium,and moderate content of Flavobacteriaceae,Pseudomonas and Achromobacter strains as well as.A small amount of Chitinophagaceae and Diaphorobacter strains were also contained.A Gram-negative strain of Diaphorobacter sp.LR2014-1 which could degrade linuron,was isolated from the microbial consortium.Strain LR2014-1 was able to efficiently hydrolyze linuron instead of mineralizing it.For the degradation dynamic of linuron,0.2 mM linuron was completely degraded in 15 h by strain LR2014-1 with an initial inoculum at OD600 of 0.1.Equivalent 3,4-dichloroaniline(3,4-DCA)was accumulated and no significant growth of strain LR2014-1 was observed.For the study of substrate spectrum of strain LR2014-1,in addition to linuron(N-methoxy-N-methyl-substituted PH),diuron,chlorotoluron,fluometuron(N,N-dimethyl-substituted PHs)and siduron the amide herbicide propanil and the carbamate herbicide swep could also be hydrolyzed.2.Classification and identification of a liniron-degradation promoting strain LIP-5TA strain named LIP-5T was isolated from the linuron degradation microbial consortium.Strain LIP-5 T could not degrade linuron or 3,4-dichloroaniline,but could grow very well by using the products or secretes during the degradation of linuron by strain LR2014-1.At the same time,strain LIP-5T can promote the degradation efficiency of linuron.On the basis of phenotypic,chemotaxonomic and phylogenetic data,strain LIP-5T was classified as representing a novel species of a new genus,for which the name Haoranjiania flava gen.nov.,sp.nov.is proposed.The type strain of Haoranjiania flava is LIP-5T(=CCTCC AB 2015365T=KCTC 42956T),to honor of the Chinese famous microbiologist Hao-Ran Jian,for the outstanding contribution he had made on microbiology.3.Cloning of the amidohydrolase gene phh and amidase gene mhhWith the high-throughput sequencing technology and combination with heterologous expression validation,two genes(phh and mhh)which encode enzymes for the hydrolysis of linuron were sucessfully cloned from strain LR2014-1.Gene phh is 1,377 bp in length,encoding 458 amino acids and has a predicted molecular weight of 49.5 kDa.Based on the amino acid sequence,Phh belongs to the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily.Phh shows the highest similarity(53%)with the thiocarbamate herbicide molinate hydrolase MolA,and followed by the two PHs amidohydrolase PuhA(51%)and PuhB(50%).Phh also has the typically His-His-Lys-His-His-Asp motif.Gene mhh is 1,425 bp in length and contains 474 amino acids and has a predicted molecular mass of 50 kDa.Mhh belongs to the amidase superfamily and contains the conserved catalytic triad(Ser-Ser-Lys)of the amidase superfamily.Mhh shows low amino acid sequence homologies with other biochemically identified amidases(27 to 38%)but 100%similarity with the newly reported triclocarban amidase TccA.The amino acid sequence similarity with LibA is only 34%.No signal peptide was found in Phh or Mhh.qRT-PCR showed that the phh and mhh genes were constitutively expressed.Meanwhile,strain LR2014-1 can form transparent halos around colony on linuron agar,which can be used as the selection marker for mini-Tn5 mutagenesis.Finaly,a mutant LR2014M with the mini-Tn5 transposon inserted into the 938-bp site of the phh gene was obtained.Compared with the substrate spectra of the wild type strain LR2014-1,the mutant LR2014M only retained the ability to degrade linuron,siduron,propanil and swep and lost the ability to degrade N,N-dimethyl-substituted PHs(fluometuron,chlortoluron and diuron).4.Characteristics of Phh and MhhThe optimal temperature and pH value of Phh for linuron hydrolysis were 35? and pH 7.5,respectively.Similar to enzymes in the metal-dependent hydrolase class,Phh was completely inhibited by 1,10-phenanthroline.The activity of Phh was dramatically inhibited by 0.2 mM and 1 mM of Zn2+or Cd2+,but slightly increased by 0.2 mM and 1 mM of Fe3+and Al3+.For Mhh,most of the tested metal ions inhibited less than 20%of its activity.The substrate spectra of Phh were linuron,fluometuron,chlortoluron and diuron.The substrate spectra of Mhh were linuron,siduron,propanil and swep,which were in line with the substrate spectra of mutant LR2014M.5.The key community structure of 3,4-dichloroaniline degrading microbial consortiumBy using the 3,4-dichloroaniline with six carbons labeled with 13C and the functional gene dcaA1 and 16S rRNA gene as the molecular markers,the key community involved in the degradation of 3,4-dichloroaniline was studied.Finaly,the functional degradation community was sucessfully labeled and separated by the ultra-high-speed density gradient centrifugation.High-throughput sequencing analysis showed that the labeled community had a significant difference with the unlabeled one.The aboundance of Proteobacteria group increased significantly in the 3 to 7 layers(the heavy layer)of labeled community,suggesting that the Proteobacteria group of bacteria played a key role in degradation of 3,4-dichloroaniline.On the genus level,the content of Diaphorobacter increased significantly in the heavy layer,the aboudance of Achromobacter and Staphylococcus increased slightly in the heavy layer,suggesting that these two genua also contributed to the degradation of 3,4-dichloroaniline.6.Isolation of 3,4-dichloroaniline degrading strains and synergistic degradation of linuron by consortiaThree strains were finally isolated from the 3,4-dichloroaniline degrading microbial consortium.The degradation efficiency of strain Achromobacter sp.ANB-1 was better than that of strain Achromobacter sp.S9-254a and strain Achromobacter sp.Amp-2,and its substrate spectrum was also much wide than that of the two strains.Strain Diaphorobacter sp.LR2014-1 and strain Achromobacter sp.ANB-1 could completely degrade linuron when they were co-cultured. |
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