摘要
3,5,6-三氯-2-吡啶醇(3,5,6-trichloro-2-pyridinol, TCP)是除草剂绿草定和杀虫剂毒死蜱及甲基毒死蜱的主要降解产物,广泛存在于环境中。在土壤中,由于TCP比毒死蜱吸附性差而稳定性和水溶性强,所以有着更强的渗透性。食入TCP对人体有危害,TCP尤其会对眼睛造成严重的损害,此外TCP对水生和陆生生态区有着低到中度的毒害作用,对生态系统影响较大。虽然通过物理和化学方法可降解TCP,但相比之下由于TCP微生物降解具有高效性,低成本,环境友好型等特点,使其在TCP环境污染治理方面更具有潜力。到目前为止,有关TCP微生物降解报道较少,因此分离和丰富TCP降解菌种资源具有十分重要的意义,本研究通过富集培养的方法分离得到一株TCP降解菌T6,然后对其进行了菌种鉴定及降解特性和降解机制研究。
从污水处理池中采得活性污泥样品,通过富集培养的方法分离得到一株稳定的3,5,6-三氯-2-吡啶醇(TCP)降解菌T6。菌株T6为一短杆状的革兰氏阴性菌,结合对菌株T6进行的16S rRNA基因序列比对和系统进化树分析,将其初步鉴定为Ralstonia sp. strain T6。
Ralstonia sp. strain T6对TCP具有高效的降解速率,能够在1h内将10mg/LTCP完全降解,在12h内将100mg/L TCP完全降解,在80h内将700mg/L TCP完全降解。Ralstonia sp. strain T6降解TCP的最适pH值和最适温度分别为8和30℃。K+对Ralstonia sp. strain T6降解TCP有促进作用；Fe3+、Ca2+、Mg2+、Mn2+对TCP降解没有明显的影响；Zn2+、Co2+、Ni2+对TCP降解有明显的抑制作用；Cu2+则完全抑制Ralstonia sp. strain T6对TCP的降解。利用硝酸银滴定法,液相色谱——质谱联机等检测手段将TCP降解过程中产生的绿色中间体初步鉴定为3,6-二羟基吡啶-2,5-二酮。
通过简并引物从Ralstonia sp. strain T6的基因组中成功扩增出利用FADH2的单加氧酶基因(tcpA)的部分序列,利用基因打靶技术将tcpA插入失活,突变菌株(T6-ΔtcpA)失去了降解TCP的能力但仍能够降解绿色中间体3,6-二羟基吡啶-2,5-二酮。推测Ralstonia sp. strain T6中参与TCP降解起始的基因位于tcpA基因序列的上下游。利用SEFA PCR技术扩增tcpA的侧翼序列,克隆到一段由3个基因组成的tcp基因簇。将tcp基因簇中的3个ORF分别命名为基因tcpR,tcpX和tcpA,长度分别为972bp,552bp和1554bp,其中TcpR和TcpX分别与LysR家族转录调节因子(LysR family transcriptional regulator)和黄素还原酶(flavin reductase)有着较高的一致性,黄素还原酶可将NAD(P)H中的H传递给FAD而生成FADH2。随后通过对突变体T6-ΔtcpA中插入失活的基因tcpA进行功能互补实验验证了互补菌株T6-ΔtcpA-com恢复了对TCP的降解功能,说明菌株T6中的tcpA基因确实为TCP降解起始阶段的关键基因,TcpA催化TCP生成绿色中间体3,6-二羟基吡啶-2,5-二酮。同时TcpA为依赖于FADH2的单加氧酶,因此推测在TcpA催化TCP降解过程中TcpX发挥着黄素还原酶的功能,为TcpA反应提供所需的FADH2,而TcpR对降解起着调节作用。
通过对菌株T6进行随机插入失活突变子文库构建,成功筛选出4个突变子,在功能上都已不能降解绿色中间体3,6-二羟基吡啶-2,5-二酮。通过侧翼序列扩增,得到了一段长约5.9kb的片断。经过序列分析,认为这一段序列中的4个基因组成了菌株T6中进一步降解绿色中间体3,6-二羟基吡啶-2,5-二酮的thp基因簇。将thp基因簇中的4个ORF分别命名为基因thpR, thpI, thpJ和thpK,长度分别为900bp,717bp,879bp和1251bp。通过构建异源重组表达载体和诱导表达的方法分别检测ThpJ和ThpI的粗酶液活性,结果发现ThpJ对绿色中间体3,6-二羟基吡啶-2,5-二酮有很好的降解活性,能将其完全降解并生成一种新的中间体5-氧基-2,4,5-三羰基正戊酸；而ThpI能将5-氨基-2,4,5-三羰基正戊酸进一步进行降解,可以看出ThpJ和ThpI能够先后对TCP降解中间体3,6-二羟基吡啶-2,5-二酮进行有效的降解。同时根据蛋白质序列同源性分析,推测ThpR和ThpK分别在thp降解基因簇中起着调节和物质运输的功能。
综上所述,菌株T6中TCP降解相关基因簇tcp基因簇和thp基因簇能够先后对TCP进行有效降解,其中tcp基因簇主要负责TCP降解初始阶段绿色中间体3,6--羟基吡啶-2,5-二酮的生成,thp基因簇能够对3,6-二羟基吡啶-2,5-二酮进行进一步的有效降解,从而实现菌株T6对TCP的彻底降解。
3,5,6-trichloro-2-pyridinol (TCP) is the main degradation product of the herbicide triclopyr and the insecticides chlorpyrifos and chlorpyrifos-methyl, and it is widespread in environment. Due to its lower sorption and greater persistence and water solubility, TCP has a much greater leaching potential than chlorpyrifos in soil. TCP is bad to human health, if swallowed, and TCP may cause serious eye damage, and it exhibits low-to-moderate toxicity to aquatic and terrestrial biota. Bioremediation is potentially a cost-effective and environment-friendly method for removal of contaminants compared to chemical and physical processes, but due to TCP's antimicrobial properties, microbial degradation has rarely been reported. A few strains that can degrade low concentrations of TCP were isolated during the screening of chlorpyrifos or chlorpyrifos-methyl degraders. The study presented here reports on the isolation and characterization of Ralstonia sp. strain T6capable of degrading TCP as a sole source of carbon and energy from activated sludge by the enrichment culture technique, and reports on the characteristics and mechanism of TCP degradation by strain T6.
A strain named T6was isolated from active sludge, which collected from a wastewater treatment system, by the enrichment culture technique. Isolate T6is a short, rod-shaped, gram-negative bacterium, and was preliminarily identified as Ralstonia sp. strain T6by analysis of its16S rRNA gene and phylogenetic tree.
Strain T6effectively metabolized10mg/L TCP in1hour,100mg/L TCP in12hours and700mg/L TCP within80hours. The optimal pH and temperature for TCP degradation by strain T6are8and30℃respectively. K+accelerated the degradation of TCP by strain T6, while Zn2+, Co2+and Ni2+seriously inhibited the degradation of TCP. Fe3+, Ca2+, Mn2+and Mg2+had no significant influence on the degradation of TCP by strain T6. Surprisingly, Cu2+completely inhibited the degradation of TCP. A green metabolite in degradation of TCP by strain T6was putatively identified as3,6-dihydroxypyridine-2,5-dione by Silver nitrate titration method and LC-MS.
A fragment of1330bp that is part of FADH2-utilizing monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6with degenerate primers. Then the tcpA gene was disrupted by homologous recombination, yielding mutant T6-ΔtcpA, which could not degrade TCP, but could degrade the green metabolite3,6-dihydroxypyridine-2,5-dione. It was deduced that tcpA and its flanking genes are related with the initiation of TCP degradation by strain T6. The flanking sequences of tcpA gene were obtained by self-formed adaptor PCR (SEFA PCR), three genes from which constitute a tcp gene cluster, and respectively named tcpR, tcpX and tcpA. The genes of tcpR, tcpX and tcpA are respectively972bp,552bp and1554bp in length, and TcpR and TcpX are respectively highly homologous to LysR family transcriptional regulator and flavin redectase that can deliver H from NAD(P)H to FAD, producing FADH2. The complementation strain T6-AtcpA-com for the disrupted tcpA gene of mutant16-ΔtcpA could degrade TCP again, indicating that tcpA is the key gene at initiation of TCP degradation by strain T6, TcpA catalyzes TCP to3,6-dihydroxypyridine-2,5-dione. Additionally, FADH2is necessary to the catalytic reaction of TcpA and flavin reductase can transfer FAD to FADH2using NAD(P)H, it is deduced that TcpX could assist TcpA by providing FADH2and TcpR plays a regulatory role in TCP degradation by strain T6.
A library of disable-degrade-green-metabolite mutants of strain T6was obtained by random mutagenesis. Four clones from6000mutants were selected, which could not degrade the green metabolite. The mutation genes and their flanking sequences were obtained by PCR amplification, four genes from the fragment of about5.9kb constitute a thp gene cluster, and respectively named thpR, thpl, thpJ and thpK. The genes of thpR, thpl, thpJ and thpK are respectively900bp,717bp,879bp and1251bp in length. The ThpI and ThpJ cell extracts from heterologous expression recombinants were assayed. ThpJ could degrade the green metabolite3,6-dihydroxypyridine-2,5-dione completely and transfer it to a new metabolite named5-amino-2,4,5-trioxopentanoic acid. ThpI could further degrade the metabolite of5-amino-2,4,5-trioxopentanoic acid. It is seen that ThpJ and Thpl collaboratively degrade the green metabolite3,6-dihydroxypyridine-2,5-dione in turn. Additionally, it is deduced that ThpR and ThpK respectively play a regulatory role and a transporter role in thp gene cluster by analysis of protein sequence homology.
In summary, the protein products of tcp and thp gene clusters from strain T6collaboratively degrade TCP in sequence, tcp gene cluster is in charge of the generation of the green metabolite3,6-dihydroxypyridine-2,5-dione at initiation of TCP degradation and thp gene cluster further degrade3,6-dihydroxypyridine-2,5-dione.
引文
[1]Anwar, S., Liaquat, F., Khan, Q.M., et al.,2009. Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by Bacillus pumilus strain C2A1[J]. J. Hazard. Mater.168(1),400-405.
[2]Armbrust, K.L.,2001. Chlorothalonil and chlorpyrifos degradation products in golf course leachate[J]. Pest Manage. Sci.57(9),797-802.
[3]Barron, M.G, Woodburn, K.B.,1995. Ecotoxicity of chlorpyrifos[J]. Rev. Environ. Contam. Toxicol.144,1-94.
[4]Baskaran, S., Kookana, R.S., Naidu, R.,2003. Contrasting behaviour of chlorpyrifos and its primary metabolite, TCP (3,5,6-trichloro-2-pyridinol), with depth in soil profiles[J]. Aust. J. Soil Res.41(4),749-760.
[5]Belchic, S.M., Xun, L.,2008. Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriavidus necator JMP134[J]. J. Bacteriol.190(5), 1615-1619.
[6]Bolognesi, C., Morasso, G.,2000. Genotoxicity of pesticides:potential risk for consumers[J]. Trends Food Sci. Technol.11,182-187.
[7]Caceres, T., He, W., Naidu, R., et al.,2007. Toxicity of chlorpyrifos and TCP alone and in combination to Daphnia carinata:The influence of microbial degradation in natural water[J]. Water Res.41,4497-4503.
[8]Cai, M., Xun, L.,2002. Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723[J]. J. Bacteriol.184,4672-4680.
[9]Chapman, R.A., Harris, C.R.,1980. Persistence of chlorpyrifos in a mineral and an organic soil[J]. J. Environ. Sci. Health B.15,39-46.
[10]Dai, M., Copley, S.D.,2004. Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723[J]. Appl. Envir. Microbiol.70,2391-2397.
[11]Egland, P.G., Gibson, J., Harwood, C.S.,2001. Reductive, coenzyme A-mediated pathway for 3-chlorobenzoate degradation in the phototrophic bacterium Rhodopseudomonas palustris[T\. Appl. Environ. Microbiol.67,1396-1399.
[12]El Fantroussi, S., Naveau, H., Agathos, S.N.,1998. Anaerobic dechlorinating bacteria[J]. Biotechnol. Prog.14,167-188.
[13]Environmental Protection Agency,1997. Review of chlorpyrifos poisoning data[C]. Environmental Protection Agency, Washington, D.C.
[14]Fang, H., Xiang, Y.Q., Hao, Y.J., et al.,2008. Fugal degradation of chlorpyrifos by V erticillium sp. DSP in pure cultures and its use in bioremediation of contaminated soil and pakchoi[J]. Int. Biodeterior. Biodegrad.61,294-303.
[15]Feng, Y.,1995. Transformation of 3,5,6-trichloro-2-pyridinol, a metabolite of pyridine e-based pesticides[D]. Ph D thesis, The Pennsylvania State University.
[16]Feng, Y., Minard, R.D., Bollag, J.M.,1998. Photolytic and microbial degradation of 3,5,6-trichloro-2-pyridinol[J]. Environ. Toxicol. Chem.17(5),814-819.
[17]Feng, Y, Racke, K.D., Bollag, J.M.,1997a. Isolation and characterization of a chlorinated-pyridinol-degrading bacterium[J]. Appl. Environ, Microbiol.63(10),4096-4098.
[18]Feng, Y, Racke, K.D., Bollag, J.M.,1997b. Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol[J]. Appl. Microbiol. Biotechnol.47(1),73-77.
[19]Fetzner, S., Lingens, F.,1994. Bacterial dehalogenases:biochemistry, genetics, and biotechnological applications [J]. Microbiol. Rev.58,641-685.
[20]Gert, W., Gabriele D.,1997. Anaerobic dehalogenases[J]. Curr. Opin. Biotechnol.8,290-295.
[21]Holliger, C., Wohlfarth, G, Diekert, G.,1999. Reductive dechlorination in the energy metabolism of anaerobic bacteria [J]. FEMS Microbiol. Rev.22,383-398.
[22]Home, I., Sutherland, T.D., Harcourt, R.L., et al.,2002. Identification of an opd(Organophosphate Degradation) gene in an Agrobacterium isolate[J]. Appl. Environ. Microbiol.68(7),3371-3376.
[23]Howard, P.H.,1991. Handbook of Environmental Fate and Exposure Data for Organic Chemicals[M]. Lewis Publishers, USA.
[24]Huang, Y, Xun, R., Chen, G, et al.,2008. Maintenance role of a glutathionyl-hydroquinone lyase (PcpF) in pentachlorophenol degradation by Sphingobium chlorophenolicum ATCC 39723[J]. J. Bacteriol.190,7595-7600.
[25]Kertesz, M.A., Cook, A.M., Leisinger, T.,1994. Microbial metabolism of sulfur-and phosphorus-containing xenobiotics[J]. FEMS Microbiol. Rev.15,195-215.
[26]Kim, J.R., Ahn, Y.J.,2009. Identification and characterization of chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol degrading Burkholderia sp. strain KR100[J]. Biodegradation 20(4),487-497.
[27]Kononova, S.V., Nesmeyanova, M.A.,2002. Phosphonates and their degradation by microorganisms[J]. Biochemistry(Moscow) 67(2),220-233.
[28]Li, K., Wang, S., Shi, Y, et al.,2011. Genome sequence of Paracoccus sp. strain TRP, a chlorpyrifos biodegrader[J]. J. Bacteriol.193(7),1786-1787.
[29]Ling, Y, Wang, H., Yong, W., et al.,2011. The effects of washing and cooking on chlorpyrifos and its toxic metabolites in vegetables[J]. Food Control 22,54-58.
[30]Li, X., He, J., Li, S.,2007. Isolation of a chlorpyrifos-degrading bacterium, Sphingomonas sp. Strain DSP-2, and cloning of the mpd gene[J]. Res. Microbiol.158,143-149.
[31]Louie, T.M., Webster, C.M., Xun, L.,2002. Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134[J]. J. Bacteriol. 184,3492-3500.
[32]Luo, L., Taylor, K.L., Xiang, H., et al.,2001. Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis[J]. Biochemistry 40,15684-15692.
[33]Macdonald, A.,1993. Performance standards for in situ bioremediation[J]. Environ.Sci. Technol.27(10),1974-1979.
[34]Manclus, J.J., Montoya, A.,1995. Development of immunossays for the analysis of chlorpyrifos and its major metabolite 3,5,6-trichloro-2-pyridinol in the aquatic environment[J]. Anal. Chim. Acta.311(3),341-348.
[35]Math, R.K., Asraful Islam, S.Md., Cho, K.M., et al.,2010. Isolation of a novel gene encoding a 3,5,6-trichloro-2-pyridinol degrading enzyme from a cow rumen metagenomic library[J]. Biodegradation 21,565-573.
[36]Maya, K., Singh, R.S., Upadhyay, S.N., et al.,2011. Kinetic analysis reveals bacterial efficacy for biodegradation of chlorpyrifos and its hydrolyzing metabolite TCP[J]. Process Biochem.46,2130-2136.
[37]Muscarella, D.E., Keown, J.F., Bloom, S.E.,1984. Evaluation of the genotoxic and embryotoxic potential of chlorpyrifos and its metabolities in vivo and in vitro[J]. Environ. Mutagen.6(1),13-23.
[38]Ni, S., Fredrickson, J.K., Xun, L.,1995. Purification and characterization of a novel 3-chlorobenzoate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1 [J].Appl. Environ. Microbiol.177,5135-5139.
[39]Orser, C.S., Dutton, J., Lange, C., et al.,1993. Characterization of a Flavobacterium glutathione S-transferase gene involved reductive dechlorination[J]. J. Bacteriol.175, 2640-2644.
[40]Padilla, L., Matus, V., Zenteno, P., et al.,2000. Degradation of 2,4,6-trichlorophenol via chlorohydroxyquinol in Ralstonia eutropha JMP134 and JMP222[J]. J. Basic Microbiol.40, 243-249.
[41]Pee, K.H., Unversucht, S.,2003. Biological dehalogenation and halogenation reactions [J]. Chemosphere 52,299-312.
[42]Petty, D.G., Getsinger, K.D., Woodburn, K.B.,2003. A review of the aquatic environmental fate of triclopyr and its major metabolites[J]. J. Aquat. Plant Manage.41,69-75.
[43]Racke, K.D.,1993. Environmental fate of chlorpyrifos. Rev. Environ[J]. Contamin. Toxicol. 131,1-154.
[44]Racke, K.D., Coats, J.R., Titus, K.R.,1988. Degradation of chlorpyrifos and its hydrolysis product,3,5,6-trichloro-2-pyridinol, in soil[J]. J. Environ. Sci. Health B.23,527-539.
[45]Racke, K.D., Laskowski, D.A., Schultz, M.R.,1990. Resistance of chlorpyrifos to enhanced biodegradation in soil[J]. J. Agric. Food. Chem.38,1430-1436.
[46]Racke, K.D., Robbins, S.T.,1991. Factors affecting the degradation of 3,5,6-trichloro-2-pyridinol in soil, in:Somasundaram, L., Coats, J.R. (Eds), Pesticide Transformation Products:Fate and Significance in the Environment[C]. (ACS Symposium Series,459) American Chemical Society, Washington, D.C., pp.93-107.
[47]Sanchez, M.A., Gonzalez, B.,2007. Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134[J]. Appl. Envir. Microbiol.73,2769-2776.
[48]Schmitz, A., Gartemarm, K.H., Fiedler, J., et al.,1992. Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU[J]. Appl. Envir. Microbiol.58,4068-4071.
[49]Scholten, J.D., Chang, K.H., Babbitt, P.C., et al.,1991. Novel enzymic hydrolytic dehalogenation of a chlorinated aromatic[J]. Science 253,182-185.
[50]Schweizer, D., Markus, A., Seez, M., et al.,1987. Purification and some properties of component B of the 4- chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS 3[J]. J. Biol. Chem.262,9340-9346.
[51]Shemer, H., Sharpless, C.M., Linden, K.G.,2005. Photodegradation of 3,5,6-trichloro-2-pyridinol in aqueous solution[J]. Water, Air, Soil Pollut.168,145-155.
[52]Singh, B.K., Walker, A., Wright, D.J., et al.,2004. Biodegradation of chlorpyrifos by Enterobacter strain B-14 and its use in bioremediation of contaminated soils[J]. Appl. Environ. Microbiol.70(8),4855-4863.
[53]Singh, D.P., Khattar, J.I.S., Nadda, J., et al.,2011. Chlorpyrifos degradation by the cyanobacterium Synechocystis sp. strain PUPCCC 64[J]. Environ. Sci. Pollut. Res.18, 1351-1359.
[54]Smidt, H., de Vos, W.M.,2004. Anaerobic microbial dehalogenation[J]. Annu. Rev.Microbiol. 58,43-73.
[55]Van der Ploeg, J., van Hall, G, Janssen, D.B.,1991. Characterization of the haloacid dehalogenase from Xanthobacter autothrophicus GJ10 and sequencing of the dhlB gene [J]. J.Bacteriol.173,7925-7933.
[56]Xu, G., Li, Y., Zheng, W., et al.,2007. Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp[J]. Biotechnol. Lett.29(10),1469-1473.
[57]Xu, GM., Zheng, W., Li, Y.Y., et al.,2008. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a newly isolated Paracoccus sp. strain TRP[J]. Int. Biodeterior. Biodegrad.62,51-56.
[58]Xun, L., Webster, C.M.,2004. A monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic Reactions[J]. J. Biol. Chem.279, 6696-6700.
[59]Yang, C., Liu, N., Guo, X., et al.,2006. Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil[J]. FEMS Microbiol. Lett.265,118-125.
[60]Yang, L., Zhao, Y.H., Zhang, B.X., et al.,2005. Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium[J]. FEMS Microbiol. Lett. 251(1),67-73.
[61]Yonezawa, Y., Urushigawa, Y.,1979. Chemico-biological interactions in biological purification system [J]. Chemosphere 8,139-142.
[62]傅国平,崔中利,吴旭平,2004.微生物降解有机磷类毒剂的酶学研究进展[J].微生物学通报3 1(2),138-143.
[63]江镇海,2009.三氯毗啶醇钠市场前景看好[J]. Shanghai Chemical Industry 34(8),36-37.
[64]孔繁翔,尹大强,严国安,2000.环境生物学[M].北京：高等教育出版社
[65]莫测辉,蔡全英,吴启堂,2001.城市污泥与玉米秸秆堆肥中多环芳烃(PAHs)的研究[J].农业工程学报17(5),73-77.
[66]张素琴,2006.微生物分子生态学[M].北京：科学出版社
[67]郑永良,陈舒丽,刘德立,2005.土壤中降解农药微生物的类别及降解特性[J].黄冈师范学院学报25(3),24-38.
[1]Adebusoye, S.A., Picardal, F.W., Ilori, M.O., et al.,2008. Characterization of multiple novel aerobic polychlorinated biphenyl (PCB)-utilizing bacterial strains indigenous to contaminated tropical African soils[J]. Biodegradation 19(1),145-159.
[2]Anwar, S., Liaquat, F., Khan, Q.M., et al.,2009. Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by Bacillus pumilus strain C2A1[J]. J. Hazard. Mater.168(1),400-405.
[3]Armbrust, K.L.,2001. Chlorothalonil and chlorpyrifos degradation products in golf course leachate[J]. Pest Manage. Sci.57(9),797-802.
[4]Barron, M.G., Woodburn, K.B.,1995. Ecotoxicity of chlorpyrifos[J]. Rev. Environ. Contam. Toxicol.144,1-94.
[5]Baskaran, S., Kookana, R.S., Naidu, R.,2003. Contrasting behaviour of chlorpyrifos and its primary metabolite, TCP (3,5,6-trichloro-2-pyridinol), with depth in soil profiles[J]. Aust. J. Soil Res.41(4),749-760.
[6]Bolognesi, C., Morasso, G.,2000. Genotoxicity of pesticides:potential risk for consumers[J]. Trends Food Sci. Technol.11,182-187.
[7]Bucheli-Witschel, M., Hafner, T., Ruegg, I., et al.,2009. Benzene degradation by Ralstonia pickettii PKO1 in the presence of the alternative substrate succinate[J]. Biodegradation 20(3), 419-431.
[8]Chapman, R.A., Harris, C.R.,1980. Persistence of chlorpyrifos in a mineral and an organic soil[J]. J. Environ. Sci. Health B.15,39-46.
[9]Chen, W.M., Chang, J.S., Wu, C.H., et al.,2004. Characterization of phenol and trichloroethene degradation by the rhizobium Ralstonia taiwanensis[J]. Res. Microbiol.155(8), 672-680.
[10]Environmental Protection Agency,1997. Review of chlorpyrifos poisoning data[C]. Environmental Protection Agency, Washington, D.C.
[11]Feng, Y,1995. Transformation of 3,5,6-trichloro-2-pyridinol, a metabolite of pyridine e-based pesticides[D]. Ph D thesis, The Pennsylvania State University.
[12]Feng, Y.C., Minard, R.D., Bollag, J.M.,1998. Photolytic and microbial degradation of 3,5,6-trichloro-2-pyridinol[J]. Environ. Toxicol. Chem.17(5),814-819.
[13]Feng, Y, Racke, K.D., Bollag, J.M.,1997. Isolation and characterization of a chlorinated-pyridinol-degrading bacterium[J]. Appl. Environ. Microbiol.63(10),4096-4098.
[14]Gu, L.F., Jiang, J.D., Li, X.H.,2007. Biodegradation of ethametsulfuron-methyl by Pseudomonas sp. SW4 isolated from contaminated soil[J]. Curr. Microbiol.55,420-426.
[15]Holt, J.G., Krieg, N.R., Sneath, P.H.A., et al.,1994. Bergey's Manual of Determinative Bacteriology,9th ed[M]. Baltimore, M.D., Lippincott Williams and Wilkins, pp.626-640.
[16]Howard, P.H.,1991. Handbook of Environmental Fate and Exposure Data for Organic Chemicals[M]. Lewis Publishers, USA.
[17]Kim, J.R., Ahn, Y.J.,2009. Identification and characterization of chlorpyrifos-methyl and 3,5,6-trichloro-2-pyridinol degrading Burkholderia sp. strain KR100[J]. Biodegradation 20(4),487-497.
[18]Ling, Y., Wang, H., Yong, W., et al.,2011. The effects of washing and cooking on chlorpyrifos and its toxic metabolites in vegetables[J]. Food Control 22,54-58.
[19]Manclus, J.J., Montoya, A.,1995. Development of immunossays for the analysis of chlorpyrifos and its major metabolite 3,5,6-trichloro-2-pyridinol in the aquatic environment[J]. Anal. Chim. Acta.311(3),341-348.
[20]Maya, K., Singh, R.S., Upadhyay, S.N., et al.,2011. Kinetic analysis reveals bacterial efficacy for biodegradation of chlorpyrifos and its hydrolyzing metabolite TCP[J]. Process Biochem.46,2130-2136.
[21]Muscarella, D.E., Keown, J.F., Bloom, S.E.,1984. Evaluation of the genotoxic and embryotoxic potential of chlorpyrifos and its metabolities in vivo and in vitro[J]. Environ. Mutagen.6(1),13-23.
[22]Petty, D.G., Getsinger, K.D., Woodburn, K.B.,2003. A review of the aquatic environmental fate of triclopyr and its major metabolites[J]. J. Aquat. Plant Manage.41,69-75.
[23]Racke, K.D.,1993. Environmental fate of chlorpyrifos. Rev. Environ[J]. Contamin. Toxicol. 131,1-154.
[24]Racke, K.D., Coats, J.R., Titus, K.R.,1988. Degradation of chlorpyrifos and its hydrolysis product,3,5,6-trichloro-2-pyridinol, in soil[J]. J. Environ. Sci. Health B.23,527-539.
[25]Racke, K.D., Laskowski, D.A., Schultz, M.R.,1990. Resistance of chlorpyrifos to enhanced biodegradation in soil[J]. J. Agric. Food. Chem.38,1430-1436.
[26]Racke, K.D., Robbins, S.T.,1991. Factors affecting the degradation of 3,5,6-trichloro-2-pyridinol in soil, in:Somasundaram, L., Coats, J.R. (Eds), Pesticide Transformation Products:Fate and Significance in the Environment[C]. (ACS Symposium Series,459) American Chemical Society, Washington, D.C., pp.93-107.
[27]Skoog, D.A., West, D.M., Holler, F.J.,1996. Fundamentals of Analytical Chemistry,7th ed[M]. Thomson Learning Inc., USA.
[28]Sudip, K.S., Bharat, B., Ashvini, C., et al.,2000. Chemotaxis of a Ralstonia sp. SJ98 toward different nitroaromatic compounds and their degradation[J]. Biochem. Biophys. Res. Commun.269(1),117-123.
[29]Trefault, N., De la Iglesia, R., Molina, A.M., et al.,2004. Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways[J]. Environ. Microbiol.6(7),655-668.
[30]Waldau, D., Mikolasch, A., Lalk, M., et al.,2009. Derivatization of bioactive carbazoles by the biphenyl-degrading bacterium Ralstonia sp. strain SBUG 290[J]. Appl. Microbiol. Biotechnol.83(1),67-75.
[31]Wang, YD., Dong, X.J., Wang, X., et al.,2007. Isolation of phenol-degrading bacteria from natural soil and their phylogenetic analysis[J]. China Environ. Sci.28(3),623-626.
[32]Xu, G, Li, Y, Zheng, W., et al.,2007. Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp[J]. Biotechnol. Lett.29(10),1469-1473.
[33]Xu, GM., Zheng, W., Li, YY, et al.,2008. Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a newly isolated Paracoccus sp. strain TRP[J]. Int. Biodeterior. Biodegrad.62,51-56.
[34]Yang, L., Zhao, Y.H., Zhang, B.X., et al.,2005. Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium[J]. FEMS Microbiol. Lett. 251(1),67-73.
[1]Belchik, S.M., Xun, L.,2008. Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriacidus necator JMP134[J]. J.Bacteriol.190(5), 1615-1619.
[2]Kitagawa, W., Miyauchi, K., Masai, E., et al.,2001. Cloning and characterization of benzoate catabolic genes in the gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1[J]. J.Bacteriol.183(22),6598-6606.
[3]Li, J., Liu, J., Shen, W., et al.,2010. Isolation and Characterization of 3,5, 6-trichloro-2-pyridinol-degrading Ralstonia sp. strain T6[J]. Bioresour. Technol.101(19), 7479-7483.
[4]Liu, Y.G, Huang, N.,1998. Eficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR[J]. Plant Mol. Biol.16, 175-181.
[5]Liu, Y.G, Mitsukawa, N., Oosumi, T., et al.,1995. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR[J]. Plant J. 8(3),457-463.
[6]Liu, Y.G, Robert, F.W.,1995. Thermal asymmetric interlaced PCR:automatable a amplification and sequencing of insert end fragment from PI and YAC clones for chromosome walking[J]. Genomics 25(3),674-681.
[7]Louie, T.M., Webster, C.M., Xun, L.,2002. Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134[J]. J.Bacteriol. 184(13),3492-3500.
[8]Matus, V., Sanchez, M.A., Martinez, M., et al.,2003. Efficient degradation of 2,4,6-trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4) [J]. Appl. Environ. Microbiol.69(12),7108-7115.
[9]Miller, S.A., Dykes, D.D., Polesky, H.F,1988. A simple salting out procedure for extracting DNA from human nucleated cells [J]. Nucleic Acids Res.16(3),1215.
[10]Qiu, J., Ma, Y, Chen, L., et al.,2011. A sirA-like gene, sirA2, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading Pseudomonas sp. HZN6 strain[J]. Appl. Microbiol. Biotechnol.92,1023-1032.
[11]Sanchez, M.A., Gonzalez, B.,2007. Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriacidus necator JMP134[J]. Appl. Environ. Microbiol.73(9),2729-2776.
[12]Wang, S.M., He, J., Cui, Z.L., et al.,2007. Self-Formed Adaptor PCR:a simple and efficient method for chromosome walking[J]. Appl. Environ. Microbiol.73(15),5048-5051.
[13]Xun, L., Sandvik, E.R.,2000. Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin Adenine dinucleotide-Utilizing monooxygenase[J]. Appl. Environ. Microbiol.66(2),481-486.
[14]Xun, L., Webster, C.M.,2004. A monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions[J]. J. Biol. Chem.279(8), 6696-6700.
[15]J.萨姆布鲁克,D.W.拉塞尔,2002.分子克隆实验指南(第三版)[M].北京：科学出版社
[16]徐冲,廖军,谢文,等,1999.链霉菌M1033同源重组葡萄糖异构酶缺陷型菌株的构建[J].科学通报44(20),2214-2219.
[1]Belchik, S.M., Xun, L.,2008. Functions of flavin reductase and quinone reductase in 2,4,6-trichlorophenol degradation by Cupriacidus necator JMP134[J]. J.Bacteriol.190(5), 1615-1619.
[2]Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem,1976,72:248-254.
[3]Feng, Y.C., Minard, R.D., Bollag, J.M.,1998. Photolytic and microbial degradation of 3,5,6-trichloro-2-pyridinol[J]. Environ. Toxicol. Chem.17(5),814-819.
[4]Li, J., Liu, J., Shen, W., et al.,2010. Isolation and Characterization of 3,5, 6-trichloro-2-pyridinol-degrading Ralstonia sp. strain T6[J]. Bioresour. Technol.101(19), 7479-7483.
[5]Li, K., Wang, S., Shi, Y., et al.,2011. Genome sequence of Paracoccus sp. strain TRP, a chlorpyrifos biodegrader[J]. J. Bacteriol.193(7),1786-1787.
[6]Louie, T.M., Webster, C.M., Xun, L.,2002. Genetic and biochemical characterization of a 2,4,6-trichlorophenol degradation pathway in Ralstonia eutropha JMP134[J]. J.Bacteriol. 184(13),3492-3500.
[7]Math, R.K., Asraful Islam, S.Md., Cho, K.M., et al.,2010. Isolation of a novel gene encoding a 3,5,6-trichloro-2-pyridinol degrading enzyme from a cow rumen metagenomic library[J]. Biodegradation 21,565-573.
[8]Matus, V., Sanchez, M.A., Martinez, M., et al.,2003. Efficient degradation of 2,4,6-trichlorophenol requires a set of catabolic genes related to tcp genes from Ralstonia eutropha JMP134(pJP4) [J]. Appl. Environ. Microbiol.69(12),7108-7115.
[9]Sanchez, M.A., Gonzalez, B.,2007. Genetic characterization of 2,4,6-trichlorophenol degradation in Cupriacidus necator JMP134[J]. Appl. Environ. Microbiol.73(9),2729-2776.
[10]Xun, L., Sandvik, E.R.,2000. Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin Adenine dinucleotide-Utilizing monooxygenase[J]. Appl. Environ. Microbiol.66(2),481-486.
[11]Xun, L., Webster, C.M.,2004. A monooxygenase catalyzes sequential dechlorinations of 2,4,6-trichlorophenol by oxidative and hydrolytic reactions[J]. J. Biol. Chem.279(8), 6696-6700.
