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1、假单胞菌WBC-3分解代谢甲基对硫磷和4-硝基酚-降解途径、调控机理及其污染的生物修复,周 宁一(Ning-Yi Zhou)中国科学院武汉病毒研究所中国科学院农业与环境微生物学重点实验室 2014年11月08日成都,第十七次全国环微会,HCB；，PCP。,第九次全国环微会（杭州,2006）代谢工程构建六氯苯降解菌,Yan DZ et al(2006)Appl.Environ.Microbiol.72:2283-2286,六氯苯,pZWXW1,Xiao Y et al(2007)J Bacteriol.189:6587-6593.,第十次全国环微会（广州,2007）2-硝基酚代谢途径,第十二次全
2、国环微会（武汉,2009）4-硝基酚代谢途径,Zhang JJ et al(2009)J Bacteriol.191:2703-2710.,Zhao et al(2009)FEMS Microbiol Ecol.70:315-323.,第十三次全国环微会（南京,2010）4-氯代硝基苯的降解机理及其污染土壤修复,第十四次全国环微会（厦门,2011）Pseudomonas putida ZWLR2-1 分解代谢2-氯硝基苯的机理研究,Liu H et al(2011)Appl.Environ.Microbiol.77:4547-4552.,第十五次全国环微会（大连,2012）微生物龙胆酸代谢途径
3、的多样性,Feng et al JBC 2006,Zhou et al.J.Bacteriol.2002,Liu and Zhou J.Bacteriol.2012,Transporters in Aromatic Compounds Catabolism,周 宁 一(Ning-Yi Zhou)中国科学院武汉病毒研究所中国科学院农业与环境微生物学重点实验室 2013年8月 兰州,微生物降解芳烃过程中转运蛋白的研究,第十六次全国环境微生物学学术研讨会兰州,Research Interests,Revealing novel microbial degradation pathways and t
4、heir molecular evolution for pollutant degradation,as well as bioremediation of contaminated soil.,中国科学院武汉病毒研究所环境微生物学科组,假单胞菌WBC-3分解代谢甲基对硫磷和4-硝基酚-降解途径、调控机理及其污染的生物修复,周 宁一(Ning-Yi Zhou)中国科学院武汉病毒研究所中国科学院农业与环境微生物学重点实验室 2014年11月08日成都,第十七次全国环微会,农药是环境污染的重要形式之一。其中，有机磷农药曾经被广泛用于世界农作物的虫害防治，曾是世界上生产和使用量最多的农药品种。如对
5、硫磷、甲基对硫磷、杀螟松、内吸磷、马拉硫磷、乐果、敌百虫及敌敌畏等。有机磷农药毒性很大，许多发达国家都已禁用或限用,且被列入PIC(Prior Informed Consent)公约。有机磷农药都含P=O或P=S基团，而这个基团可以通过水解反应而被分解。,有机磷农药降解示意图。R是烷基、芳基、烃基或其他基团，X是卤素、脂肪酸、芳香族或是杂环基团。OPH:organophosphate hydrolase encoded by oph,opd,or opdA gene,(Serdar et al.,1982;Harper et al.,1988;Serdar et al.,1985;Horne
6、et al.,2002;Sogorb&Vilanova,2002),甲基对硫磷降解菌株的筛选,甲基对硫磷（Methyl Parathion,MP）是有机磷农药的代表，曾被大量用于农业生产中的害虫防治，但其残留会造成环境与食品的严重污染,严重影响了人类的身体健康。对于甲基对硫磷污染物的生物修复是一种经济和有效的方法。,甲基对硫磷,Rani&Lalithakumari,1994,Cui,Z.L.,S.P.Li&G.P.Fu,Appl.Environ.Microb.,2001,67:4922-4925.,Methyl Parathion Hydrolase(mpd)in Plesiomonas sp
7、.M6,甲基对硫磷降解菌株的筛选,从湖北沙市地区的农药污染土样中被分离出来一株能够彻底降解甲基对硫磷的Pseudomonas sp.WBC-3菌株。该菌株能以甲基对硫磷（MP）和4-硝基酚（PNP）为唯一碳源、氮源和能源。,陈亚丽等.2002.微生物学报.42:490-497,假单胞菌WBC-3电镜照片,甲基对硫磷,WBC-3,甲基对硫磷降解菌株的筛选,The catabolic genes for MP and PNP degradation in Pseudomonas sp.WBC-3 were located on a large lasmid(70Kb).,FIG.A.Plasmid
8、 isolated from WBC-3 and its derivatives.Lane 1,markers;lane 2,plasmid of WBC-3;lane 3,plasmid of transconjugant WXP-6;lane 4,plasmid of PaW340;lane 5,plasmid of cured strain WBC-C.B.Restriction enzyme analysis of plasmids from WBC-3 and transconjugant WXP-6.Degradation of PNP by Pseudomonas sp.stra
9、in WBC-3(C)and transconjugant WXP-6(D).,A,B,C,Liu H,JJ Zhang,SJ Wang,XE Zhang,and NY Zhou.2005.Biochem.Biophys.Res.Commun.334:1107,WBC-3,WXP-6,D,WBC-3菌株中分解代谢甲基对硫磷的机理,MPH and OPD demonstrate 12%identity at amino acid level,Diversity of Organophosphate Hydrolases,WBC-3菌株中分解代谢甲基对硫磷的机理,Harper et al.,198
10、8;Mulbry Liu et al.,2005,FIG.A.Replicative transposition of Tnmphfrom pZWWM002.B.Southern blot hybridization analysis of the end products of Tnmph transposition in Pseudomonas putida PaW340.,A transposable class I composite transposon carrying mph(methylparathion hydrolase)from Strain WBC-3(Wei et a
11、l.,2009),A,B,WBC-3菌株中分解代谢甲基对硫磷的机理,Figure 1.A.(b)A cartoon representation of the monomer structure of MPH.(c and d)The MPH dimer.B.(a)A stereo Figure showing electron density covering the metal center.(d)The active site of MPH,showing the metal binding center.,Dong et al.,J Mol Biol,2005.,Crystal str
12、ucture of methyl parathion hydrolase from Strain WBC-3,A,B,WBC-3菌株中分解代谢甲基对硫磷的机理,？,WBC-3,MPH,WBC-3菌株中分解代谢甲基对硫磷的机理,FIG.1.(A)Proposed pathway for methyl parathion and PNP catabolism in Pseudomonas sp.strain WBC-3.(B)Organization of the pnp gene cluster of strain WBC-3.,MPH,The pathway for methyl parath
13、ion and PNP catabolism in Pseudomonas sp.strain WBC-3,Zhang JJ,H Liu,Y Xiao,XE Zhang,NY Zhou.2009.J.Bacteriol.191:2703-2710.,PnpA,PnpB,PnpCD,PnpE,PnpF,WBC-3菌株中分解代谢4-硝基酚的机理,20/21,FIG.A.SDS-PAGE of H6-PnpA and H6-PnpB.B.The reactions catalyzed by pnpA gene product and the proposed pathway for PNP cata
14、bolism.C.Spectrophotometric changes during the transformation of PNP by purified H6-PnpA.,Zhang JJ,H Liu,Y Xiao,XE Zhang,NY Zhou.2009.J.Bacteriol.191:2703-2710.,A,B,C,PnpA catalyzes monooxygenation of PNP to p-benzoquinone,WBC-3菌株中分解代谢4-硝基酚的机理,FIG.A.对苯二醌在还原酶（PnpB）的作用下还原为对苯二酚;B.pnpB是菌株WBC-3降解4-硝基酚的必需
15、基因，pnpB的缺陷型菌株利用4-硝基酚的能力被显著抑制。,PnpB catalyzes reduction of p-benzoquinone to hydroquinone,A,B,Zhang JJ,H Liu,Y Xiao,XE Zhang,NY Zhou.2009.J.Bacteriol.191:2703-2710.,WBC-3菌株中分解代谢4-硝基酚的机理,g-HMSA,22/28,HQ,PnpCD，PnpE catalyzes reaction of hydroquinone to maleyacetate,WBC-3菌株中分解代谢4-硝基酚的机理,PnpCD,PnpE,B.Pnp
16、CD,C.PnpE,FIG.A.The reactions catalyzed by pnpA gene product and the proposed pathway for PNP catabolism.Spectrophotometric changes during the transformation of hydroquinone(HQ)by purified H6-PnpCD(B)and the transformation of g-hydroxymuconic semialdehyde by purified H6-PnpE(C).,A,23/32,PnpA,PnpB,Pn
17、pCD,PnpE,PnpF,PnpA,PnpG,The pathway for PNP and 4-NC catabolism in P.sp.strain WBC-3,FIG.(A)Proposed pathway for PNP and 4-NC catabolism in Pseudomonas sp.strain WBC-3.(B)Organization of the pnp gene cluster of strain WBC-3.,A,B,(Wei et al.,2010,21(6):915),WBC-3菌株中分解代谢4-硝基邻苯二酚的机理,FIG.A.Degradation o
18、f PNP bystrain WBC-3 cells incubated with PNP or 4-NC.B.Degradation of 4-NC bystrain WBC-3 cells incubated with PNP or 4-NC.C.Time course of 4-NC transformation by resting E.coli cells pZWJJ011 expressing PnpA.(Wei et al.,2010,21(6):915),Degradation of 4-nitrocatechol(4-NC)by strain WBC-3 incubated
19、with PNP,C,WBC-3菌株中分解代谢4-硝基邻苯二酚的机理,FIG.Transformation of 4-NC by purified H6-PnpA.(Wei et al.,2010,21(6):915),PnpA catalyzes monooxygenation of 4-NC to hydroxyquinol,WBC-3菌株中分解代谢4-硝基邻苯二酚的机理,FIG.A.SDS-PAGEwith Coomassie Blue staining of H6-PnpG.B.Enzymatic oxidation of hydroxyquinol by purified H6-Pn
20、pG,PnpG is a hydroxyquinol 1,2-dioxygenase,lmax=290nm,lmax=245nm,A,B,(Wei et al.,2010,21(6):915),PnpG,WBC-3菌株中分解代谢4-硝基邻苯二酚的机理,PnpA,PnpB,PnpCD,PnpE,PnpF,PnpA,PnpG,The pathway for PNP and 4-NC catabolism in P.sp.strain WBC-3,FIG.(A)Proposed pathway for PNP and 4-NC catabolism in Pseudomonas sp.strain
21、WBC-3.(B)Organization of the pnp gene cluster of strain WBC-3.,A,B,(Wei et al.,2010,21(6):915),WBC-3菌株中分解代谢4-硝基邻苯二酚的机理,WBC-3菌株中4-硝基酚分解代谢途径的调控,FIG.1.(A)Proposed pathway for methyl parathion and PNP catabolism in Pseudomonas sp.strain WBC-3.(B)Organization of the pnp gene cluster of strain WBC-3.,The
22、pathway for methyl parathion and PNP catabolism in Pseudomonas sp.strain WBC-3,Zhang JJ,H Liu,Y Xiao,XE Zhang,NY Zhou.2009.J.Bacteriol.191:2703-2710.,orf6,WBC-3菌株中4-硝基酚分解代谢途径的调控,A LysR-like regulatory protein PnpR was found from genome sequencing of strain WBC-3,FIG.A.Four ellipses stand for PnpR te
23、tramer.The curved and dotted arrow stands for binding of para-nitrophenol(PNP)with PnpR.Four thin arrows stand for PnpRs activating four operons of pnpA,pnpB,pnpCDEFG and pnpR.Two forward slashes stands for the gap in genome.B.Transcriptional organization of genes pnpC,D,E,F and G.,Zhang WM,JJ Zhang
24、,X Jiang,HJ Chao,and NY Zhou.Appl.Environ.Microbiol.2014.,WBC-3菌株中4-硝基酚分解代谢途径的调控,FIG.Transcriptional analysis of pnpA,pnpB,pnpCDEFG and pnpR operons in wild type strain WBC-3(A),pnpR knockout strain WBC3-pnpR(B)and complemented pnpR-knockout strain WBC3-pnpRC(C),in the presence and absence of PNP.D.
25、The cultures grow at 30 for 52 h in PNP+MM.,D,pnpR gene is essential for utilization PNP and pnp gene cluster transcription,(Zhang et al.,2014.),WBC-3菌株中4-硝基酚分解代谢途径的调控,Zhang WM,JJ Zhang,X Jiang,HJ Chao,and NY Zhou.Appl.Environ.Microbiol.2014.,WBC-3菌株中4-硝基酚分解代谢途径的调控,FIG.A.DNase I footprinting analysi
26、s of PnpR binding to the sense strand of PpnpAB.B.DNase I footprinting analysis of PnpR binding to the antisense strand of PpnpAB.,pnpR binds with the promoter regions of pnpA-pnpB,pnpC and pnpR,Weblogo of motif(PnpR binding with pnp promoters),PnpR与pnp启动子 的调控结合位点（RBS）为 GTT-N11-AAC，与LysR家族调控蛋白的调控结合位
27、点(T-N11-A)相符合。,Zhang et al.Appl.Environ.Microbiol.2014.,WBC-3菌株中4-硝基酚分解代谢途径的调控,Motif required for high-affinity PnpR binding and activation of pnpA,pnpB,pnpCDEFG and pnpR promoters,甲基对硫以及硝基酚污染土壤的生物修复,FIG.Chemical analysis of degradation of MP and PNP.(A)Removal of MP in different soil microcosms;(B)
28、PNP removal or release in different soil microcosms.(C)Nitrite release in different soil microcosms.:Native soil(T1);:Native soil+MP(T2);:Native soil+MP+WBC-3(T3);:Sterile soil+MP(T4);:Sterile soil+MP+WBC-3(T5);:Native soil+PNP(T6).(Wang et al.,2014),假单胞菌WBC-3对甲基对硫磷污染的土壤进行生物修复，15天内能将甲基对硫磷完全降解，无有毒中间体
29、残留。,甲基对硫磷污染土壤的生物修复,硝基酚微生物代谢的途径,Xiao et al,2007,J.BacteriolZhang et al,2009,J.BacteriolXiao et al,2012,J.Bacteriol,三种硝基酚同分异构体污染土壤的生物修复,Pseudomonas sp.strain WBC-3，Cupriavidus necator JMP134 和Alcaligenes sp.strain NyZ215的混合菌群对三种硝基同分异构体污染土壤的生物修复.(a)对硝基酚生物降解;(b)间硝基酚降解;(c)邻硝基酚降解;(d)生物修复过程中NO2-浓度的变化(e)NH4
30、+浓度的变化.,文章：Chi XQ.Zhou NY.2013.Environ Pollut.172:33-41,PNP,MNP,ONP,NH4+,NO2-,NS：自然土壤SS：灭菌土壤,NPs:三种硝基酚（PNP,MNP,ONP）,consortium:三种接入菌(WBC-3,JMP134,NyZ215),三种硝基酚同分异构体污染土壤的生物修复,Chi XQ,JJ Zhang,S Zhao and NY Zhou.2013.Environ.Pollut.172:33-41,假单胞菌WBC-3分解代谢甲基对硫磷和4-硝基酚,Pseudomonas sp.WBC-3,The history of the lab in Wuhan,谢谢,41/32,
