谷胱甘肽转移酶PcpF对Sphingobium chlorophenolicum ATCC 39723中五氯苯酚（PCP）降解途径的修护作用的研究
摘要
五氯苯酚(PCP)是一种人工合成的多氯化芳香化合物,作为木材防腐剂的一种主要成分,它是世界上最为通用的一种杀虫剂和杀真菌剂,自二十世纪三十年代开始被大规模的投入商业生产与使用。现在自然界的水体,大气与土壤中都可以检测到PCP的存在,因此,PCP已成为一种主要的环境污染物质。PCP可与细胞膜相结合,从而破坏细胞正常生长所需物质的跨膜转运,同时也会阻碍能量的生成。短时间大剂量的接触PCP,可引起人体高烧,大量出汗,行动失调,肌肉抽搐,以至昏迷;长时间的接触PCP,其毒性可造成对肝脏,肾脏,皮肤,血液,神经系统,生殖系统,和胃肠道的损害,甚至导致速死。
已报道的可降解五氯苯酚的微生物都是通过对PCP污染土质的菌种筛选获得,其中S.Chlorophenolicum ATCC 39723是1985年由美国明尼苏达州被PCP污染的土壤中筛选发现的。已有的研究结果表明,参与S.ChlorophenolicumATCC 39723降解PCP代谢途径的酶主要包括PcpA,PcpB,PcpC,PcpE.另外一个调节蛋白PcpR参与相关编码基因簇的转录调控。
2,3,5,6-四氯对羟基苯酚还原去卤化酶(PcpC)是一种zeta型谷胱甘肽转移酶。在PCP降解过程中,PcpC可将2,3,5,6-四氯对羟基苯酚(TeCH)降解为2,3,6-三氯-对羟基苯酚(TriCH),进而进一步降解为2,6-二氯-对羟基苯酚(DiCH)。研究发现PcpC在纯化过程中极易被氧化失活,同时在催化TeCH和TriCH转化过程中生成谷胱甘肽-2,3,6-三氯-对羟基苯酚(GS-TriCH)和谷胱甘肽-2,6-二氯-对羟基苯酚(GS-DiCH)复合物,而GS-TriCH与GS-DiCH的转化过程还不清楚。通过对Sphingobium Chlorophenolicum ATCC 39723中PCP降解基因簇的进一步分析发现,在PCP下游紧邻的开放阅读框19(orf19)也编码一个谷胱甘肽转移酶,将其命名为PcpF。本论文通过对PcpF的克隆、表达、纯化和性质研究,分析了PcpF在PCP降解途径中对PcpC功能所起到的维护和补充作用,进一步明确完善了S.Chlorophenolieum ATCC 39723对PCP的降解途径和降解机制。
本论文首先将PcpF在大肠杆菌中进行了诱导过量表达,表达的PcpF经过细胞破碎、硫酸铵沉淀,苯基-琼脂糖分子筛柱层析和DEAE-琼脂糖阴离子交换柱层析等4个分离纯化步骤,达到电泳纯。通过对PcpF的活性测定发现,PcpF可将氧化态PcpC降解TeCH产生的GS-TriCH复合物进一步降解为DiCH。在氧化态PcpC和PcpF的联合反应中,两种酶的混合物可以将TeCH完全降解为DiCH,PcpF对GS-TriCH的专一性酶活为288±46 nmol min~(-1)mg~(-1)。对PepF进行的底物专一性试验结果表明,PcpF在有还原力NADPH存在时,可作用于脱氢抗坏血酸和β-羟已基-二硫代(β-hydroxyethyl disulfide),其比活分别为440±90 nmol min~(-1)mg~(-1),353±14 nmol min~(-1) mg~(-1)。以脱氢抗坏血酸为底物,测得PcpF的最适作用pH值为pH 7.2-8.0,最适作用温度为40℃,最适作用离子强度为20 mM。
PcpC和PcpF序列中各自都有两个半胱氨酸基团。前期研究结果显示PepC的N-末端半胱氨酸Cys14位于其活性位点内,一旦将Cys14突变,突变体PcpC将丧失部分活性,只能将TeCH转化为GS-DiCH和GS-TriCH复合物。对PcpF中相应的两个半胱氨酸分别进行点突变,结果表明,N-末端半胱氨酸Cys53突变后,PcpF完全丧失活性,而Cys248的突变对PcpF活性无明显影响。半胱氨酸突变的实验结果表明,PcpC和PepF都可以被氧化破坏而丧失活性。用不同浓度的过氧化氢(H_2O_2)分别处理PcpC和PcpF,将部分氧化失活的PcpC单独作用于TeCH,同时将部分氧化失活的PcpF的与过量的完全氧化失活的PcpC混合后共同作用于TeCH,结果显示,PcpC在6.4mM H_2O_2处理后丧失95%的活力,而PcpF在相同处理条件下仍保持40%的活力。实验结果表明,在处于细胞内相同氧化能力的情况下,PcpC氧化后,PcpF仍可以将氧化态PcpC降解转化TeCH产生的GS-DiCH和GS-TriCH复合物进一步降解为DiCH。
在S.Chlorophenolicum ATCC 39723中,通过插入失活的方法将pcpF敲除。对敲除后的突变株与野生型S.Chlorophenolicum ATCC 39723降解PCP的能力进行了比较。实验结果显示,在液体培养状态下,当有其他碳源存在时,野生株与突变株都可在40 min作用完全降解100μM PCP;而当以PCP作为唯一碳源时,野生株S.Chlorophenolicum ATCC 39723完全降解100μM PCP需要两个小时,而突变株在完全相同的培养条件下,降解100μM PCP需要四个小时。其中可能的原因在于碳源充足的情况下,细胞生长旺盛,细胞内有大量还原力存在,PcpC不易被氧化,因此低浓度的PCP(100μM)在野生株和突变株中的降解差别不大;而当碳源缺乏、细胞生长停滞的时候,细胞内积累的氧自由基增多,PcpC在细胞中极易被氧化,因此野生株和pcpF-缺失突变株对100μM PCP的降解能力相差一倍。在固体平板培养状态下,野生株在含有400μM PCP的平板上仍显示旺盛生长,而pcpF-突变株只在100μM PCP的平板上显示微弱生长。这些数据表明,一旦pcpF被敲除,氧化后的PcpC降解TeCH生成的GS-DiCH和GS-TriCH复合物在细胞内累积,从而对细胞产生毒性,抑制细胞生长。
本论文还对来自Mesorhizobium sp.BNC1,Cupriavidus.necator JMP134的两个与PcpF序列相似性超过50%的谷胱甘肽转移酶进行了克隆表达、纯化和活性研究。实验结果显示,来自Mesorhizobium sp.BNC1和Cupriavidus.necalorJMP134的两个谷胱甘肽转移酶也都具有转化GS-DiCH和GS-TriCH复合物的能力。同时还发现,大肠杆菌的细胞破碎液也可以作用于TeCH,生成GS-DiCH和GS-TriCH复合物。对来自大肠杆菌MG1655的一个谷胱甘肽转移酶和8个推测的谷胱甘肽转移酶也进行了研究,通过对其中六个基因gst,yliJ,yibF,yqjG,yfcF,sspA的克隆和产物表达,以及对其中七个基因突变株gst-,yliJ-,yqjG-,yfcF-,sspA-,yghU-,yfcG-细胞破碎液的活性研究并没有发现其降解转化TeCH的活力相对于野生菌株有明显的升高或者降低。但这些实验数据表明,微生物中存在的谷胱甘肽转移酶普遍具有通过转移谷胱甘肽基团降解多氯化芳香化合物的作用,可由此进行对谷胱甘肽转移酶活性功能进行进一步的研究。
Pentachlorophenol(PCP) is a toxic pollutant.Its biodegradation has been extensively studied in Sphingobium chlorophenolicum ATCC 39723.All enzymes required to convert PCP to a common metabolic intermediate before entering the tricarboxylic acid cycle for complete mineralization have been characterized.One of the enzymes is tetrachloro-p-quinol reductive dehalogenase(PcpC),which is a glutathione S-transferase(GST).PcpC catalyzes the GSH-dependent conversion of tetrachloro-p-quinol to trichloro-p-quinol(TriCH) and then to dichloro-p-quinol (DiCH) in the PCP degradation pathway.PcpC is readily oxidatively damaged,and the damaged PcpC produces glutathionyl GS-TriCH and GS-DiCH conjugates,which cannot be further metabolized by the enzyme.Accumulation of GS-quinol conjugates inside the cells could be detrimental to the cells,as the conjugates could undergo oxidation and reduction,generating reactive oxygen species.A hypothetical GST gene (pcpF) is next to pcpC on the bacterial chromosome.The pcpF gene was cloned,and PcpF was purified.The purified PcpF was able to convert GS-TriCH and GS-DiCH conjugates to TriCH and DiCH with the co-consumption of GSH.The GS-quinol lyase reactions catalyzed by PcpF are rather unusual for a GST.The inactivation of pcpF in S.chlorophenolicum made the mutant Jose the GS-quinol lyase activities.The mutant became more sensitive to PCP toxicity and significantly decreased the PCP degradation rates.Thus,PcpF played a maintenance role in PCP degradation and converted GS-quinol conjugates back to the intermediates of PCP degradation pathway.
已报道的可降解五氯苯酚的微生物都是通过对PCP污染土质的菌种筛选获得,其中S.Chlorophenolicum ATCC 39723是1985年由美国明尼苏达州被PCP污染的土壤中筛选发现的。已有的研究结果表明,参与S.ChlorophenolicumATCC 39723降解PCP代谢途径的酶主要包括PcpA,PcpB,PcpC,PcpE.另外一个调节蛋白PcpR参与相关编码基因簇的转录调控。
2,3,5,6-四氯对羟基苯酚还原去卤化酶(PcpC)是一种zeta型谷胱甘肽转移酶。在PCP降解过程中,PcpC可将2,3,5,6-四氯对羟基苯酚(TeCH)降解为2,3,6-三氯-对羟基苯酚(TriCH),进而进一步降解为2,6-二氯-对羟基苯酚(DiCH)。研究发现PcpC在纯化过程中极易被氧化失活,同时在催化TeCH和TriCH转化过程中生成谷胱甘肽-2,3,6-三氯-对羟基苯酚(GS-TriCH)和谷胱甘肽-2,6-二氯-对羟基苯酚(GS-DiCH)复合物,而GS-TriCH与GS-DiCH的转化过程还不清楚。通过对Sphingobium Chlorophenolicum ATCC 39723中PCP降解基因簇的进一步分析发现,在PCP下游紧邻的开放阅读框19(orf19)也编码一个谷胱甘肽转移酶,将其命名为PcpF。本论文通过对PcpF的克隆、表达、纯化和性质研究,分析了PcpF在PCP降解途径中对PcpC功能所起到的维护和补充作用,进一步明确完善了S.Chlorophenolieum ATCC 39723对PCP的降解途径和降解机制。
本论文首先将PcpF在大肠杆菌中进行了诱导过量表达,表达的PcpF经过细胞破碎、硫酸铵沉淀,苯基-琼脂糖分子筛柱层析和DEAE-琼脂糖阴离子交换柱层析等4个分离纯化步骤,达到电泳纯。通过对PcpF的活性测定发现,PcpF可将氧化态PcpC降解TeCH产生的GS-TriCH复合物进一步降解为DiCH。在氧化态PcpC和PcpF的联合反应中,两种酶的混合物可以将TeCH完全降解为DiCH,PcpF对GS-TriCH的专一性酶活为288±46 nmol min~(-1)mg~(-1)。对PepF进行的底物专一性试验结果表明,PcpF在有还原力NADPH存在时,可作用于脱氢抗坏血酸和β-羟已基-二硫代(β-hydroxyethyl disulfide),其比活分别为440±90 nmol min~(-1)mg~(-1),353±14 nmol min~(-1) mg~(-1)。以脱氢抗坏血酸为底物,测得PcpF的最适作用pH值为pH 7.2-8.0,最适作用温度为40℃,最适作用离子强度为20 mM。
PcpC和PcpF序列中各自都有两个半胱氨酸基团。前期研究结果显示PepC的N-末端半胱氨酸Cys14位于其活性位点内,一旦将Cys14突变,突变体PcpC将丧失部分活性,只能将TeCH转化为GS-DiCH和GS-TriCH复合物。对PcpF中相应的两个半胱氨酸分别进行点突变,结果表明,N-末端半胱氨酸Cys53突变后,PcpF完全丧失活性,而Cys248的突变对PcpF活性无明显影响。半胱氨酸突变的实验结果表明,PcpC和PepF都可以被氧化破坏而丧失活性。用不同浓度的过氧化氢(H_2O_2)分别处理PcpC和PcpF,将部分氧化失活的PcpC单独作用于TeCH,同时将部分氧化失活的PcpF的与过量的完全氧化失活的PcpC混合后共同作用于TeCH,结果显示,PcpC在6.4mM H_2O_2处理后丧失95%的活力,而PcpF在相同处理条件下仍保持40%的活力。实验结果表明,在处于细胞内相同氧化能力的情况下,PcpC氧化后,PcpF仍可以将氧化态PcpC降解转化TeCH产生的GS-DiCH和GS-TriCH复合物进一步降解为DiCH。
在S.Chlorophenolicum ATCC 39723中,通过插入失活的方法将pcpF敲除。对敲除后的突变株与野生型S.Chlorophenolicum ATCC 39723降解PCP的能力进行了比较。实验结果显示,在液体培养状态下,当有其他碳源存在时,野生株与突变株都可在40 min作用完全降解100μM PCP;而当以PCP作为唯一碳源时,野生株S.Chlorophenolicum ATCC 39723完全降解100μM PCP需要两个小时,而突变株在完全相同的培养条件下,降解100μM PCP需要四个小时。其中可能的原因在于碳源充足的情况下,细胞生长旺盛,细胞内有大量还原力存在,PcpC不易被氧化,因此低浓度的PCP(100μM)在野生株和突变株中的降解差别不大;而当碳源缺乏、细胞生长停滞的时候,细胞内积累的氧自由基增多,PcpC在细胞中极易被氧化,因此野生株和pcpF-缺失突变株对100μM PCP的降解能力相差一倍。在固体平板培养状态下,野生株在含有400μM PCP的平板上仍显示旺盛生长,而pcpF-突变株只在100μM PCP的平板上显示微弱生长。这些数据表明,一旦pcpF被敲除,氧化后的PcpC降解TeCH生成的GS-DiCH和GS-TriCH复合物在细胞内累积,从而对细胞产生毒性,抑制细胞生长。
本论文还对来自Mesorhizobium sp.BNC1,Cupriavidus.necator JMP134的两个与PcpF序列相似性超过50%的谷胱甘肽转移酶进行了克隆表达、纯化和活性研究。实验结果显示,来自Mesorhizobium sp.BNC1和Cupriavidus.necalorJMP134的两个谷胱甘肽转移酶也都具有转化GS-DiCH和GS-TriCH复合物的能力。同时还发现,大肠杆菌的细胞破碎液也可以作用于TeCH,生成GS-DiCH和GS-TriCH复合物。对来自大肠杆菌MG1655的一个谷胱甘肽转移酶和8个推测的谷胱甘肽转移酶也进行了研究,通过对其中六个基因gst,yliJ,yibF,yqjG,yfcF,sspA的克隆和产物表达,以及对其中七个基因突变株gst-,yliJ-,yqjG-,yfcF-,sspA-,yghU-,yfcG-细胞破碎液的活性研究并没有发现其降解转化TeCH的活力相对于野生菌株有明显的升高或者降低。但这些实验数据表明,微生物中存在的谷胱甘肽转移酶普遍具有通过转移谷胱甘肽基团降解多氯化芳香化合物的作用,可由此进行对谷胱甘肽转移酶活性功能进行进一步的研究。
Pentachlorophenol(PCP) is a toxic pollutant.Its biodegradation has been extensively studied in Sphingobium chlorophenolicum ATCC 39723.All enzymes required to convert PCP to a common metabolic intermediate before entering the tricarboxylic acid cycle for complete mineralization have been characterized.One of the enzymes is tetrachloro-p-quinol reductive dehalogenase(PcpC),which is a glutathione S-transferase(GST).PcpC catalyzes the GSH-dependent conversion of tetrachloro-p-quinol to trichloro-p-quinol(TriCH) and then to dichloro-p-quinol (DiCH) in the PCP degradation pathway.PcpC is readily oxidatively damaged,and the damaged PcpC produces glutathionyl GS-TriCH and GS-DiCH conjugates,which cannot be further metabolized by the enzyme.Accumulation of GS-quinol conjugates inside the cells could be detrimental to the cells,as the conjugates could undergo oxidation and reduction,generating reactive oxygen species.A hypothetical GST gene (pcpF) is next to pcpC on the bacterial chromosome.The pcpF gene was cloned,and PcpF was purified.The purified PcpF was able to convert GS-TriCH and GS-DiCH conjugates to TriCH and DiCH with the co-consumption of GSH.The GS-quinol lyase reactions catalyzed by PcpF are rather unusual for a GST.The inactivation of pcpF in S.chlorophenolicum made the mutant Jose the GS-quinol lyase activities.The mutant became more sensitive to PCP toxicity and significantly decreased the PCP degradation rates.Thus,PcpF played a maintenance role in PCP degradation and converted GS-quinol conjugates back to the intermediates of PCP degradation pathway.
引文
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24. Watanabe, I. 1973 Isolation of pentachlorophenol-decomposing bacteria from soil. Soil Sci.Plant Nutr. 19:109-116
25. Resnick, S. M., and P. J. Chapman. 1994. Physiological properties and substrate specificity of a pentachlorophenol-degrading Pseudomonas species. Biodegradation 5(1): 47-54.
26. Radehaus, P. M., and S. K. Schmidi. 1992. characterization of a novel Pseudomonas sp.that mineralizes high concentrations of pentachlorophenol. Appl. Environ. Microbiol.58:2879-2885
27. Crawford, R. L., and M. M. Ederer. 1999. Phylogeny of Sphingomonas species that degrade pentachlorophenol. J. Ind. Microbiol. Biotechnol. 23(4-5) :320-325
28. Ederer, M. M., R. L.Crawford, R. P. Herwig, and C. S. Orser. 1996. PCP degradation is mediated by closely related strains of the genus Sphingomonas. Mol Ecol 6(1): 39-49
29. Mikesell, M. D., and S. A. Boyd. 1986. Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Appl Environ Microbiol.52(4): 861-865
30. Ornston, L. N., and E. I. Neidle. 1991. Evolution of genes for the b-ketoadipate pathway in Acinetobacter calcoaceticus. Plenum Press, New York.
31. Cserjesi, A. J. 1967. The adaptation of fungi to pentachlorophenol and its biodegradation.Can. J. Microbiol. 13:1243-1249.
32. Karlson, U., F. Rojo, J. D. van Elsas, and E. Moore. 1995. Genetic and serological evidence for the recognition of four pentachlorophenol-degrading becteria strains as a species of the genus Sphingomonas. Syst. Appl. Microbiol. 18: 539-548.
33. Nohynek, L. J., E. L. Suhonen, E. L. Nurmiaho-Lassila, J. Hantula, and M.Salkinoja-Salenen. 1995. Description of four pentachlorophenol-degrading bacteria strains of sphingomonas chlorophenolica sp. nov. Syst. Appl. Mcirobiol. 18:527-538.
34. Oser, C. S., C. C. lange, L. Xun, T.C. Zahrt, and B. J. Schneider. 1993. Cloning, sequence analysis, and expression of the Flavobacterium pentachlorophenol 4-monooxygenase gene in E.coli. J. Bacteriol. 175:411-416.
35. Xun, L., and C. S. Orser. 1991. Purification and properties of pentachlorophenol hydroxylase: a flavoprotein from Flavobacterium sp. strain ATCC39723. J. Bacteriol.173:4447-4453.
36. Dai, M., J. B. Rogers, J. R. Warner, and S. D. Copley. 2003. A previously unrecognized step in pentachlorophenol degradation in Sphingobium chlowphenolicum is catalyzed by tetrachlorobenzoquinone reductase (PcpD). J. Bacteriol. 185:302-310
37. Cai, M., and L. Xun. 2002. Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723. J. Bacteriol. 184:4672-4680.
38. Xun, L., E. Topp, and C.S. Orser 1992. Glutathione is the reducing agent for reductive dehlogenation of tetrachloro-p-hydroquinone by extracts from a Flavobacterium sp. Biochem.Biophy. Res. Comm. 182:361-366.
39. Xun, L., E. Topp, and C.S. Orser. 1992. purification and characterization of a tetrachloro-hydroquinone reductive dehalogenase from a Flavobacterium sp. J. Bacteriol.174:8003-8007.
40. Ohtsubo, Y., K. Miyauchi, K. Kanda, T. Hatta, H. Kiyohara, T. Senda, Y. Nagata, Y.Mitsui, and M. Takagi. 1999. PcpA, which is involved in the degradation of Pentachlorophenol in Sphingomonas chlorophenolica ATCC 39723, is a novel type of ring-cleavage dioxygenase. FEBS Lett. 459:395-398.
41. Orser, C. S., J. Dutton, C. Lange, P. jablonski, L. Xun, and M. Hargis. 1993b.characterization of a Flavobacterium glutathione S-transferase gene involved in reductive dechlorination. J. Bacteriol. 175: 2640-2644.
42. Sitting, M. 1981. Handbook of toxic and hazardous chemicals. Noyes publications., Park Ridge, NJ.
43. Barbeau, C, L. Deschenes, D.karamanev, Y. Comeau, and R. Samson. 1997.Bioremediation of Pentachlorophenol-contaminated soil by bioaugmentation using activated soil. Appl Microbiol Biotechnol 48(6): 745-752.
44. Beaudet, R., M. J. Levesque, R. villemur, M. Lanthier, M. Chenier, F. Lepine, and J. G.Bisaillon. 1998. Anaerobic biodegradation of Pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Dsulfitobacterium frappieri strain PCP-1.Appl Microbiol Biotechnol 50 (1): 135-141.
45. Colores, G M., P. M. Radehaus, and S. K. Schmidt. 1995. Use of a Pentachlorophenol degrading bacterium to bioremediate highly contaminated soil. Appl. Biochem Biotechnol 54(1-3): 271-275.
46. Mannisto, M. K., M. S. Salkinoja-salonen, and J. A. Puhakka. 2001. Insitu polychlorophenol bioremediation potential of the indigenous bacterial community of boreal groundwater. Water Res 35(10):2496-2504.
47. McCarthy, D. L., S. Navarrete, W. S. Willett, P. C. Babbitt, and S. D. Copley. 1996.Exploration of the Relationship between Tetrachlorohydroquinone Dehalogenase and the Glutathione S-Transferase Superfamily. Biochem. 35:14634-14642.
48. Sheehan, D., G. Meade, V. M. Foley, and C. A. Dowd. 2001 Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360:1-16.
49. Masai, E., A. Ichimura, Y. Sato, K. Miyauchi, Y. Katayama, and M. Fukuda. 2003. Roles of the enantioselective glutathione S-transferases in cleavage of β-aryl ether. 185:1768-1775.
50. Bolton, J, L., M. A. Trush, T. M. Penning, G Dryhurst, and T. J. Monks. 2000. Role of quinones in toxicology. Chem. Res. Toxicol. 13:135-160.
51. Xun, L., J. Bohuslavek, and M. Cai. 1999. Characterization of 2,6-dichlorop-hydroquinone 1,2-dioxygenase (PcpA) of Sphingomonas chlorophenolica ATCC 39723. Biochem. Biophy.Res. Comm. 266:322-325.
52. Myers, E. W., and W. Miller. 1988. Optimal alignments in linear space. Comput. Applic.Biosci. 4:11-17.
53. Board, P. G, M. Coggan, G Chelvanayagam, S. Easteal, L. S. Jermiin, G K. Schulte, D.E. Danley, L. R. Hoth, M. C. Griffor, A. V. Kamath, M. H. Rosner, B. A. Chrunyk, D. E.Perregaux, C. A. Gabel, K. F. Geoghegan, and J. Pandit. 2000. Identification,characterization, and crystal structure of the Omega class glutathione transferases. J. Biol.Chem. 275:24798-24806
54. Saber, D. L., and R. L. Crawford. 1985. Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol. Appl. Environ. Microbiol. 50:1512-1518
55. Xun, L., E. Topp, and C. S. Orser. 1992. Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp. J. Bacteriol.174:8003-8007.
56. Datsenko, K. A., Wanner, B. L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 97: 6640-6645
57. Nishida M, Kong KH, Inoue H, Takahashi K. 1994 Molecular cloning and site-directed mutagenesis of glutathione S-transferase from Escherichia coli. The conserved tyrosyl residue near the N terminus is not essential for catalysis. J Biol Chem. 269: 32536-32541
58. Hansen AM, Qiu Y, Yeh N, Blattner FR, Durfee T, Jin DJ. 2005 SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol Microbiol.56:719-734
59. Kanai T, Takahashi K, Inoue H. 2006. Three distinct-type glutathione S-transferases from Escherichia coli important for defense against oxidative stress. J Biochem. 140:703-711.
60. Nortemann, B. 1992. Total degradation of EDTA by mixed cultures and a bacterial isolate.Appl. Environ. Microbiol. 58:671-676.
61. Louie, T. M.., C. M. Webster, and L. Xun. 2002. Genetic and Biochemical Characterization of a 2,4,6-TrichIorophenol Degradation Pathway in Ralstonia eutropha JMP134. J. Bacteriol.184:3492-3500.
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11. Chhabra, R. S., R. M. Maronpot, J. R. Bcher, J. K. Haseman, J. D. Toft, and M. R.Hejtmancik. 1999. Toxicology and carcinogenesis studies of pentachlorophenol in rats.Toxicol. Sci. 48: 14-20.
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14. Apajalahti, J.H.A., P. Karpanoja, and M. S. Salkinoja-salonen. 1986. Rhodococcus chlorophenolicus sp. Nov., a chlorophenol-mineralizing actinomycete. Int. J. Syst. Bacteriol.36:246-251
15. Chu, J. P., and E. J. Kirsch. 1972. Metabolismof pentachlorophenol by an axenic bacterial culture. Appl. Microbiol. 23:1033-1035.
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19. Lee, S. G, B. D. Yoon, Y. H. Park, and H. M. Oh. 1988. Isolation of a novel pentachlorophenol-degrading bacterium, Pseudomonas sp. Bu34. J Appl Microbiol 85: 1-8
20. Saber, D. L., and C. R. L. 1985. Isolation and characterization of Flavobacterium strains that degraded pentachlorophenol. Appl. Environ. Microbiol. 50:1512-1518
21. Schenk, T., R. Muller, M.K. Morsberger, K. Otta, and F. lingens. 1989. Enzymatic dehalogenation of pentachlorophenol by extracts from Arthrobacter sp. Strain ATCC 33790. J.Bacteriol. 171:5487-5491
22. Stanlake, G J., and R. K. Finn. 1982. Isolation and characterization of a pentachlorophenol-degrading bacterium. App. Environ. Microbiol. 44:1421-1427
23. Suzuki, T. 1977. Metabolism of pentachlorophenol by a soil microbe. J. Environ. Sci. Health Part B 12:113-127
24. Watanabe, I. 1973 Isolation of pentachlorophenol-decomposing bacteria from soil. Soil Sci.Plant Nutr. 19:109-116
25. Resnick, S. M., and P. J. Chapman. 1994. Physiological properties and substrate specificity of a pentachlorophenol-degrading Pseudomonas species. Biodegradation 5(1): 47-54.
26. Radehaus, P. M., and S. K. Schmidi. 1992. characterization of a novel Pseudomonas sp.that mineralizes high concentrations of pentachlorophenol. Appl. Environ. Microbiol.58:2879-2885
27. Crawford, R. L., and M. M. Ederer. 1999. Phylogeny of Sphingomonas species that degrade pentachlorophenol. J. Ind. Microbiol. Biotechnol. 23(4-5) :320-325
28. Ederer, M. M., R. L.Crawford, R. P. Herwig, and C. S. Orser. 1996. PCP degradation is mediated by closely related strains of the genus Sphingomonas. Mol Ecol 6(1): 39-49
29. Mikesell, M. D., and S. A. Boyd. 1986. Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Appl Environ Microbiol.52(4): 861-865
30. Ornston, L. N., and E. I. Neidle. 1991. Evolution of genes for the b-ketoadipate pathway in Acinetobacter calcoaceticus. Plenum Press, New York.
31. Cserjesi, A. J. 1967. The adaptation of fungi to pentachlorophenol and its biodegradation.Can. J. Microbiol. 13:1243-1249.
32. Karlson, U., F. Rojo, J. D. van Elsas, and E. Moore. 1995. Genetic and serological evidence for the recognition of four pentachlorophenol-degrading becteria strains as a species of the genus Sphingomonas. Syst. Appl. Microbiol. 18: 539-548.
33. Nohynek, L. J., E. L. Suhonen, E. L. Nurmiaho-Lassila, J. Hantula, and M.Salkinoja-Salenen. 1995. Description of four pentachlorophenol-degrading bacteria strains of sphingomonas chlorophenolica sp. nov. Syst. Appl. Mcirobiol. 18:527-538.
34. Oser, C. S., C. C. lange, L. Xun, T.C. Zahrt, and B. J. Schneider. 1993. Cloning, sequence analysis, and expression of the Flavobacterium pentachlorophenol 4-monooxygenase gene in E.coli. J. Bacteriol. 175:411-416.
35. Xun, L., and C. S. Orser. 1991. Purification and properties of pentachlorophenol hydroxylase: a flavoprotein from Flavobacterium sp. strain ATCC39723. J. Bacteriol.173:4447-4453.
36. Dai, M., J. B. Rogers, J. R. Warner, and S. D. Copley. 2003. A previously unrecognized step in pentachlorophenol degradation in Sphingobium chlowphenolicum is catalyzed by tetrachlorobenzoquinone reductase (PcpD). J. Bacteriol. 185:302-310
37. Cai, M., and L. Xun. 2002. Organization and Regulation of Pentachlorophenol-Degrading Genes in Sphingobium chlorophenolicum ATCC 39723. J. Bacteriol. 184:4672-4680.
38. Xun, L., E. Topp, and C.S. Orser 1992. Glutathione is the reducing agent for reductive dehlogenation of tetrachloro-p-hydroquinone by extracts from a Flavobacterium sp. Biochem.Biophy. Res. Comm. 182:361-366.
39. Xun, L., E. Topp, and C.S. Orser. 1992. purification and characterization of a tetrachloro-hydroquinone reductive dehalogenase from a Flavobacterium sp. J. Bacteriol.174:8003-8007.
40. Ohtsubo, Y., K. Miyauchi, K. Kanda, T. Hatta, H. Kiyohara, T. Senda, Y. Nagata, Y.Mitsui, and M. Takagi. 1999. PcpA, which is involved in the degradation of Pentachlorophenol in Sphingomonas chlorophenolica ATCC 39723, is a novel type of ring-cleavage dioxygenase. FEBS Lett. 459:395-398.
41. Orser, C. S., J. Dutton, C. Lange, P. jablonski, L. Xun, and M. Hargis. 1993b.characterization of a Flavobacterium glutathione S-transferase gene involved in reductive dechlorination. J. Bacteriol. 175: 2640-2644.
42. Sitting, M. 1981. Handbook of toxic and hazardous chemicals. Noyes publications., Park Ridge, NJ.
43. Barbeau, C, L. Deschenes, D.karamanev, Y. Comeau, and R. Samson. 1997.Bioremediation of Pentachlorophenol-contaminated soil by bioaugmentation using activated soil. Appl Microbiol Biotechnol 48(6): 745-752.
44. Beaudet, R., M. J. Levesque, R. villemur, M. Lanthier, M. Chenier, F. Lepine, and J. G.Bisaillon. 1998. Anaerobic biodegradation of Pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Dsulfitobacterium frappieri strain PCP-1.Appl Microbiol Biotechnol 50 (1): 135-141.
45. Colores, G M., P. M. Radehaus, and S. K. Schmidt. 1995. Use of a Pentachlorophenol degrading bacterium to bioremediate highly contaminated soil. Appl. Biochem Biotechnol 54(1-3): 271-275.
46. Mannisto, M. K., M. S. Salkinoja-salonen, and J. A. Puhakka. 2001. Insitu polychlorophenol bioremediation potential of the indigenous bacterial community of boreal groundwater. Water Res 35(10):2496-2504.
47. McCarthy, D. L., S. Navarrete, W. S. Willett, P. C. Babbitt, and S. D. Copley. 1996.Exploration of the Relationship between Tetrachlorohydroquinone Dehalogenase and the Glutathione S-Transferase Superfamily. Biochem. 35:14634-14642.
48. Sheehan, D., G. Meade, V. M. Foley, and C. A. Dowd. 2001 Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J. 360:1-16.
49. Masai, E., A. Ichimura, Y. Sato, K. Miyauchi, Y. Katayama, and M. Fukuda. 2003. Roles of the enantioselective glutathione S-transferases in cleavage of β-aryl ether. 185:1768-1775.
50. Bolton, J, L., M. A. Trush, T. M. Penning, G Dryhurst, and T. J. Monks. 2000. Role of quinones in toxicology. Chem. Res. Toxicol. 13:135-160.
51. Xun, L., J. Bohuslavek, and M. Cai. 1999. Characterization of 2,6-dichlorop-hydroquinone 1,2-dioxygenase (PcpA) of Sphingomonas chlorophenolica ATCC 39723. Biochem. Biophy.Res. Comm. 266:322-325.
52. Myers, E. W., and W. Miller. 1988. Optimal alignments in linear space. Comput. Applic.Biosci. 4:11-17.
53. Board, P. G, M. Coggan, G Chelvanayagam, S. Easteal, L. S. Jermiin, G K. Schulte, D.E. Danley, L. R. Hoth, M. C. Griffor, A. V. Kamath, M. H. Rosner, B. A. Chrunyk, D. E.Perregaux, C. A. Gabel, K. F. Geoghegan, and J. Pandit. 2000. Identification,characterization, and crystal structure of the Omega class glutathione transferases. J. Biol.Chem. 275:24798-24806
54. Saber, D. L., and R. L. Crawford. 1985. Isolation and characterization of Flavobacterium strains that degrade pentachlorophenol. Appl. Environ. Microbiol. 50:1512-1518
55. Xun, L., E. Topp, and C. S. Orser. 1992. Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp. J. Bacteriol.174:8003-8007.
56. Datsenko, K. A., Wanner, B. L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 97: 6640-6645
57. Nishida M, Kong KH, Inoue H, Takahashi K. 1994 Molecular cloning and site-directed mutagenesis of glutathione S-transferase from Escherichia coli. The conserved tyrosyl residue near the N terminus is not essential for catalysis. J Biol Chem. 269: 32536-32541
58. Hansen AM, Qiu Y, Yeh N, Blattner FR, Durfee T, Jin DJ. 2005 SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol Microbiol.56:719-734
59. Kanai T, Takahashi K, Inoue H. 2006. Three distinct-type glutathione S-transferases from Escherichia coli important for defense against oxidative stress. J Biochem. 140:703-711.
60. Nortemann, B. 1992. Total degradation of EDTA by mixed cultures and a bacterial isolate.Appl. Environ. Microbiol. 58:671-676.
61. Louie, T. M.., C. M. Webster, and L. Xun. 2002. Genetic and Biochemical Characterization of a 2,4,6-TrichIorophenol Degradation Pathway in Ralstonia eutropha JMP134. J. Bacteriol.184:3492-3500.