恶臭假单胞菌CD2 P型ATP酶基因cadA1抗Zn~（2+）功能的确定
|
摘要
基因cadA1编码的是P型ATP酶，它是一种能够对细胞内重金属离子起到平衡调节作用的运输子。但是在恶臭假单胞菌(Pseudomonas putida)中，cadA1在抗重金属方面的功能还知道的很少。本研究的目的就是为了确定cadA1是否与P.putida CD2的重金属抗性相关。
本研究构建了P.putida CD2的cadA1中断突变株，然后通过测定该突变株对不同重金属的抗性能力来研究cadA1的功能，将中断突变株对Cd~(2+)、Zn~(2+)、Co~(2+)、Pb~(2+)、Cu~(2+)和Mn~(2+)6种重金属的最小抑制浓度(MIC)进行了测定。结果显示，与野生型相比，MIC值并未有所变化。这表明了cadA1中断突变株与野生型对这6种重金属的抗性能力不存在差异。
为了确定cadA1是否与重金属的抗性有关，也将cadA1在对重金属敏感的P.putida CD2突变株中进行了过量表达，观察细菌对重金属的抗性水平是否有提高。结果显示cadA1的过量表达能够提高P.putida CD2的czcCBA1突变株对Zn~(2+)的抗性能力。这说明了CadA1能够提供对Zn~(2+)的抗性，但这样一个结论却没有在cadA1的中断实验中得到证实。这给我们的启示是：cadA1在P.putida CD2中确实是有抗重金属的功能，但是它可能受到了细胞内某些因子的调控从而不能施展其自身的能力。
本研究还利用绿色荧光蛋白测定的方法来初步研究了cadA1的表达调控。通过构建基因报告质粒pEGP-PCAD，然后利用它来监测恶臭假单胞菌中(野生型菌株，czcCBA1突变株，cadA2突变株)重金属对cadA1启动子的表达量的影响。结果显示cadA1启动子的表达只能够在P.putida CD2-2(也就是czcCBA1突变株)中被诱导，而且只被Zn~(2+)诱导。这些也就说明了在P.putida CD2中，cadA1的表达只能被Zn~(2+)诱导，而且CzcCBA1的存在会抑制这个诱导作用。
结合上述三个方面的结果，最后确定了cadA1的功能：cadA1能够提供对Zn~(2+)的抗性，但是它只是在P.putida CD2中，当czcCBA1发生了突变时才会大量合成蛋白CadA1，以此来作为细胞中的一个补救途径抵抗Zn~(2+)。在一般情况下，由于CzcCBA1的存在，CadA1的量非常之小以至于它在细胞对重金属的解毒中根本起不到任何作用。据我们了解，本研究是第一次证明了在P.putida CD2中P型ATP酶基因cadA1与Zn~(2+)的抗性有关。
The cadA1 gene encodes a P-type ATPase in P. putida. The P-type ATPases are a family of transporters that mediate the uptake and extrusion of heavy metal ions. But little is known about the function of cadA1 in heavy metal resistance. The aim of the present work is to determine if the cadA1 gene is involved in tolerance to heavy metal ions in P. putida CD2.
In this study, a mutant strain of P. putida CD2 was constructed by disrupting cadA1. The function of cadA1 was studied by determining the resistance of the cadA1 disrupted strain to different heavy metal ions. But no difference in the MIC was observed between P. putida CD2-1 and CD2. It was shown that the cadA1 disrupted strain did not change the ability in resistance to Cd~(2+)、Zn~(2+)、Co~(2+)、Pb~(2+)、Cu~(2+) and Mn~(2+) compared with the wild type.
For determining the effect of CadA1 on heavy metal resistance, the cadA1 gene was A1so overexpressed in heavy metal-sensitive mutants of P. putida CD2. The overexpression of cadA1 increased resistance to Zn~(2+) in the czcCBA1 disrupted mutant of P. putida CD2. It indicates that the cadA1 gene confers Zn~(2+) resistance which has not been proven by disrupting cadA1, suggesting that the cadA1 gene is functional, but may be regulated by some unknow factors in P. putida CD2 that it cannot exert its function in heavy metal resistance.
Further more, the regulation of cadA1 expression was studied by GFP fluorescence assay. A reporter plasmid pEGP-PCAD was constructed and used to monitor the expression of PcadA1 in response to heavy metal ions in P. putida CD2、CD2-2 and CD2-3. It showed that PcadA1 was specific, responding at a significant level to Zn~(2+) in P. putida CD2-2. But there was A1most no expression of PcadA1 in P. putida CD2 or CD2-3 when induced by heavy metal ions. It reveals that expression of cadA1 is induced by Zn~(2+) and this induction is inhibited by the presence of CzcCBA1 in P. putida CD2.
In combination of the results obtained, it was demonstrated that cadA1 can confer resistance to Zn~(2+), but the level of CadA1 in P. putida CD2 is so low because of the presence of CzcCBAl that it contributes little to Zn~(2+) resistance. However, expression of cadA1 would increase to serve as a salvage pathway for Zn~(2+) resistance when czcCBA1 disrupted. To the best of our knowledge, this work has provided the first evidence that the cadA1 gene confers Zn~(2+) resistance in P. putida CD2.
本研究构建了P.putida CD2的cadA1中断突变株，然后通过测定该突变株对不同重金属的抗性能力来研究cadA1的功能，将中断突变株对Cd~(2+)、Zn~(2+)、Co~(2+)、Pb~(2+)、Cu~(2+)和Mn~(2+)6种重金属的最小抑制浓度(MIC)进行了测定。结果显示，与野生型相比，MIC值并未有所变化。这表明了cadA1中断突变株与野生型对这6种重金属的抗性能力不存在差异。
为了确定cadA1是否与重金属的抗性有关，也将cadA1在对重金属敏感的P.putida CD2突变株中进行了过量表达，观察细菌对重金属的抗性水平是否有提高。结果显示cadA1的过量表达能够提高P.putida CD2的czcCBA1突变株对Zn~(2+)的抗性能力。这说明了CadA1能够提供对Zn~(2+)的抗性，但这样一个结论却没有在cadA1的中断实验中得到证实。这给我们的启示是：cadA1在P.putida CD2中确实是有抗重金属的功能，但是它可能受到了细胞内某些因子的调控从而不能施展其自身的能力。
本研究还利用绿色荧光蛋白测定的方法来初步研究了cadA1的表达调控。通过构建基因报告质粒pEGP-PCAD，然后利用它来监测恶臭假单胞菌中(野生型菌株，czcCBA1突变株，cadA2突变株)重金属对cadA1启动子的表达量的影响。结果显示cadA1启动子的表达只能够在P.putida CD2-2(也就是czcCBA1突变株)中被诱导，而且只被Zn~(2+)诱导。这些也就说明了在P.putida CD2中，cadA1的表达只能被Zn~(2+)诱导，而且CzcCBA1的存在会抑制这个诱导作用。
结合上述三个方面的结果，最后确定了cadA1的功能：cadA1能够提供对Zn~(2+)的抗性，但是它只是在P.putida CD2中，当czcCBA1发生了突变时才会大量合成蛋白CadA1，以此来作为细胞中的一个补救途径抵抗Zn~(2+)。在一般情况下，由于CzcCBA1的存在，CadA1的量非常之小以至于它在细胞对重金属的解毒中根本起不到任何作用。据我们了解，本研究是第一次证明了在P.putida CD2中P型ATP酶基因cadA1与Zn~(2+)的抗性有关。
The cadA1 gene encodes a P-type ATPase in P. putida. The P-type ATPases are a family of transporters that mediate the uptake and extrusion of heavy metal ions. But little is known about the function of cadA1 in heavy metal resistance. The aim of the present work is to determine if the cadA1 gene is involved in tolerance to heavy metal ions in P. putida CD2.
In this study, a mutant strain of P. putida CD2 was constructed by disrupting cadA1. The function of cadA1 was studied by determining the resistance of the cadA1 disrupted strain to different heavy metal ions. But no difference in the MIC was observed between P. putida CD2-1 and CD2. It was shown that the cadA1 disrupted strain did not change the ability in resistance to Cd~(2+)、Zn~(2+)、Co~(2+)、Pb~(2+)、Cu~(2+) and Mn~(2+) compared with the wild type.
For determining the effect of CadA1 on heavy metal resistance, the cadA1 gene was A1so overexpressed in heavy metal-sensitive mutants of P. putida CD2. The overexpression of cadA1 increased resistance to Zn~(2+) in the czcCBA1 disrupted mutant of P. putida CD2. It indicates that the cadA1 gene confers Zn~(2+) resistance which has not been proven by disrupting cadA1, suggesting that the cadA1 gene is functional, but may be regulated by some unknow factors in P. putida CD2 that it cannot exert its function in heavy metal resistance.
Further more, the regulation of cadA1 expression was studied by GFP fluorescence assay. A reporter plasmid pEGP-PCAD was constructed and used to monitor the expression of PcadA1 in response to heavy metal ions in P. putida CD2、CD2-2 and CD2-3. It showed that PcadA1 was specific, responding at a significant level to Zn~(2+) in P. putida CD2-2. But there was A1most no expression of PcadA1 in P. putida CD2 or CD2-3 when induced by heavy metal ions. It reveals that expression of cadA1 is induced by Zn~(2+) and this induction is inhibited by the presence of CzcCBA1 in P. putida CD2.
In combination of the results obtained, it was demonstrated that cadA1 can confer resistance to Zn~(2+), but the level of CadA1 in P. putida CD2 is so low because of the presence of CzcCBAl that it contributes little to Zn~(2+) resistance. However, expression of cadA1 would increase to serve as a salvage pathway for Zn~(2+) resistance when czcCBA1 disrupted. To the best of our knowledge, this work has provided the first evidence that the cadA1 gene confers Zn~(2+) resistance in P. putida CD2.
引文
1． 陈素华，孙铁，周启星．微生物与重金属之间的相互作用及其应用研究．应用生 态学报，2002，13(2)：239-242
2． 陈玉成．污染环境生物修复工程．北京：化学工业出版社，2003
3． 胡南，赵斌．一株耐镉恶臭假单胞菌的分离鉴定及其重要的镉抗性相关基因的克 隆．[硕士学位论文]．武汉：华中农业大学，2006
4． 贾广宁．重金属污染的危害与防治．有色矿冶，2004，20(1)：39-42
5．李洪亮，陈玉成．污水处理中重金属的毒理学研究进展．微量元素与健康研究， 2006，23(1)：49-53
6． 田建民．用微生物外红硫螺菌属形成的生物聚合物去除废水中的重金属．太原 理工大学学报，1999，30(2)：175-178
7． 王保军，杨惠芳．微生物与重金属的相互作用．重庆环境科学，1996，18(1)： 35-38
8． 卫扬保．微生物生理学．高等教育出版社，1989
9． 谢曙光．重金属对生物除磷影响的研究．[硕士学位论文]．西安理工大学，2000
10．叶锦韶，尹华，彭辉．微生物抗重金属毒性研究进展．环境污染治理技术与设备， 2002，3(4)：1-4
11.Alonso A, Sanchez P, et al. Stenotrophomonas maltophilia D457R contains a cluster of genes from gram-positive bacteria involved in antibiotic and heavy metal resistance. Antimicrob Agents Chemother, 2000, 44(7): 1778-1782
12. Anton A, Grosse C, et al. CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34. J Bacteriol, 1999, 181(22): 6876-6881
13. Banjerdkij P, Vattanaviboon P, Mongkolsuk S. Exposure to cadmium elevates expression of genes in the OxyR and OhrR regulons and induces cross-resistance to peroxide killing treatment in Xanthomonas campestris. Appl Environ Microbiol, 2005, 71: 1843-1849
14. Berson O, Lidstrom M E. Cloning and characterization of corA, a gene encoding a copper-repressible polypeptide in the type I methanotroph, Methylomicrobium albus BG8. FEMS Microbiol Lett, 1997, 148(2): 169-174
15. Bertini I, Hartmann H J, Klein T, et al. High resolution solution structure of the protein part of Cu7 metallothionein. Eur J Biochem, 2000, 267 (4): 1008-1018
16. Blattner F R, Plunkett G R, Bloch C A. The complete genome sequence of Escherichia coli K-12. Science, 1997, 277: 1453-1474
17. Bloss T, Clemens S, et al. Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes. Planta, 2002, 214(5): 783-791
18. Borremans B, Hobman J L, et al. Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Barteriol, 2001, 183 (19): 5651-5658
19. Caccavo F J. Protein-mediated adhesion of the dissimilatory Fe( III )-reducing bacterium Shewanella alga BrY to hydrous ferric oxide. Appl Environ Microbiol, 1999,65:5017-5022.
20. Canovas D, Cases I, de Lorenzo V. Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis. Environ Microbiol, 2003, 5: 1242-1256
21. Claudia G, Mark K, et al. Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem, 2000, 275(41): 32310-32316
22. Coyle P, Philcox J C, et al. Metallothionein: the multipurpose protein. Cell Mol Life Sci, 2002, 59(4): 627-47
23. Dey S, Papadopoulou B, Haimeur A, Roy G, Grondin K, Dou D, Rosen B P, Ouellette M. High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system. Mol Biochem Parasitol, 1994, 67: 49-57
24. Dey S, Rosen B P. Dual mode of energy coupling by the oxyanion-translocating ArsB protein. J Bacteriol, 1995, 1770: 385-389
25. Dopson M, Baker-Austin C, Koppineedi P R, Bond P L. Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic micro-organisms. Microbiology, 2003, 149(8): 1959-1970
26. Dopson M, Lindstrom E B. Potential role of thiobacillus caldus in arsenopyrite bioleaching. Appl Environ Microbiol, 1999, 65(11): 36-40
27. Eccles H. Treatment of metal-contaminated wastes: why select a biological process? Trends Biotechnol, 1999, 17(12): 462-465
28. Endo G, Silver S. CadC, the transcriptional regulatory protein of the cadmium resistance system of S taphylococcus aureus plasmid pI258. J Bacteriol, 1995, 177(15): 4437-4441
29. Fagan M J, Saier M H J. P-typ ATPases of eukaryotes and bacteria: sequence comparisons and construction of phylogenetic trees. J Mol Evol, 1994, 38: 57-99
30. Gadd G M. Bioremedial potential of microbiol mechanisms of metal mobilization and immobilization. Curr Opin Biotechnol, 2000, 11: 271-279.
31. Gonzalez-Guerrero M, Azcon-Aguilar C, et al. Characterization of a Glomus intraradices gene encoding a putative Zn transporter of the cation diffusion facilitator family. Fungal Genet Biol, 2005, 42(2): 130-40.
32. Grosse C, Anton A, et al. Identification of a regulatory pathway that controls the heavy-metal resistance system Czc via promoter czcNp in Ralstonia metallidurans. Arch Microbiol, 2004, 182(2-3): 109-118
33. Gross C, Kelleher M, Iyer V R, et al. Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem, 2000, 275(41): 32310-32316
34. Hao Z, Chen S, Wilson D B. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum. Appl Environ M icrobiol, 1999, 65(11): 4746-4752
35. Haney C J, Grass G, et al. New developments in the understanding of the cation diffusion facilitator family. J Ind Microbiol Biotechnol, 2005, 32(6): 215-226
36. Hassan M T, van der Lelie D, Springael D, Romling U, Ahmed N, Mergeay M. Identification of a gene cluster, czr, involved in cadmium and zinc resistance in pseudomonas aeruginosa. Gene, 1999, 238: 417-425
37. Heaton A C, Rugh C L, Kim T, et al. Toward detoxifying mercury-polluted aquatic sediments with rice genetically engineered for mercury resistance. Environ Toxicol Chem, 2003, 22(12): 2940-2947
38. Hernandez A, Mellado R P, Martinez J L. Metal accumulation and vanadium -induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae. Appl Environ Microbiol, 1998, 64(11): 4317-4320
39. Horitsu H, Yamamoto K, Wachi S, Kawai K, Fukuchi A. Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil. J Bacteriol, 1986,165:334-335
40. Hu N, Zhao B. Key genes involved in heavy-metal resistance in Pseudomonas putida CD2. FEMS Microbiol Lett, 2007, 267: 17-22
41. Inoue Y, Kobayashi S, Kimura A. Cloning and phenotypic characterization of a gene enhancing resistance against oxidative stress in Saccharomyces cerevisiae. J Ferment Bioeng, 1993, 75:327-331
42. Jin Y H, Clark A B, Slebos R J, Al-Refai H, Taylor J A, Kunkel T A, Resnick M A, Gordenin D A. Cadmium is a mutagen that acts by inhibiting mismatch repair. Nat Genet, 2003, 34: 326-329
43. Jonathan J, Alice L, et al. Cd( II)-responsive and constitutive mutants implicate a novel domain in MerR. J Bacteriol, 1999, 181(11): 3462-3471
44. Kagi J H. Overview of metallothionein. Methods Enzymol, 1991, 205: 613-626
45. Kambe T, Suzuki T, et al. Sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters. Genomics Proteomics Bioinformatics, 2006, 4(1): 1-9
46. Kannan G M, Tripathi N, et al. Toxic effects of arsenic (III) on some hematopoietic and central nervous system variables in rats and guinea pigs. J Toxicol Clin Toxicol, 2001, 39(7): 675-682
47. Khunajakr N, Liu C Q, Charoenchai P. A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis. Gene, 1999, 229: 229-235
48. Kim D, Gustin J L, et al. The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae. Plant J, 2004, 39(2): 237-251
49. Kurdihaidar B, Horn D K, Flittner D E, Heath D, Fink L, Naredi P, Howell S B. Dual cytoplasmic and nuclear distribution of the novel arsenite-stimulated human ATPase (hASNA-I). J Cell Biochem, 1998,71: 1-10
50. Kuyucak N, Volesky B. Biosorbents for recovery of metals from industrial solutions. Biotechnol Let, 1988, 10(2): 137-142
51. Lee S W, Glickmann E, Cooksey D A. Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator. Appl Environ Microbiol, 2001, 67: 1437-1444
52. Legatzki A, Grass G, Anton A, Rensing C, Nies D H. Interplay of the Czc system and two P-type ATPases in conferring metal resistance to Ralstonia metallidurans. J Bacteriol, 2003, 185: 4354-4361
53. Legatzki A, Franke S, et al. First step towards a quantitative model describing Czc-mediated heavy metal resistance in Ralstonia metallidurans. Biodegradation, 2003, 14(2): 153-168
54. Leedjarv A, Ivask A, Virta M. The interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440. J Bacteriol, 2007, doi:10.1128/JB.01494-07
55. Liu J, Gladysheva T B, Lee L, Rosen B P. Identification of an essential cysteinyl residue in the ArsC arsenate reductase of plasmid 8773. Biochemistry, 1995, 34: 13472-13476
56. Liu J, Rosen B P. Ligand interactions of the ArsC arsenate reductase. J Biol Chem, 1997,272:21084-21089
57. Lloyd J R. Microbial reduction of metals and radionuclides. FEMS Microbiol Rev, 2003, 27(2-3): 411-425
58. Lunin V V, Dobrovetsky E, et al. Crystal structure of the CorA Mg2+ transporter. Nature, 2006, 440(7085): 833-837
59. Maguire M E. The structure of CorA: a Mg2+-selective channel. Curr Opin Struct Biol, 2006, 16(4): 432-438
60. Mergeay M, Nies D, et al. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol, 1985, 162(1): 328-334
61. Mejare M, Bulow L. Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends Biotechnol, 2001, 19(2): 67-73
62. Miyabe S, Izawa S, et al. Expression of ZRC1 coding for suppressor of zinc toxicity is induced by zinc-starvation stress in Zap1-dependent fashion in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 2000, 276(3): 879-884
63. Misra T K.Bacterial resistance to inorganic mercucy salts and organomercurials. Plasmid, 1992,27:4-16
64. Moncrief M B, Maguire M E. Magnesium transport in prokaryotes. J Biol Inorg Chem, 1999, 4(5): 523-527
65. Navarre C, Wu L F, Mandrand B M A. The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel. Mol Microbiol, 1993,9:1181-1191
66. Nelson D L, Kennedy E P. Magnesium transport in Escherichia coli. Inhibition by cobaltous ion. J Biol Chem, 1971, 246(9): 3042-3049
67. Nies D H. CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus. J Bacteriol, 1992, 174(24): 8102-8110
68. Nies D H. Microbiol heavy-metal resistance. Appl Microbiol Biotechnol, 1999, 51(6): 730-750
69. Nies D H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev, 2003, 27: 313-339
70. Nies D H, Silver S. Ion efflux systems involved in bacterial metal resistances. J Ind Microbiol, 1995, 14(2): 186-199
71. Pardo R, Herguedas M, Barrado E, Vega M. Biosorption of cadmium, copper, lead and zinc by inactive biomass of Pseudomonas Putida. Anal Bioanal Chem, 2003, 376: 26-32
72. Park M H, Wong B B, Lusk J E. Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology. J Bacteriol, 1976, 126(3): 1096-1103
73. Patrick L. Toxic metals and antioxidants: Part II. The role of antioxidants in arsenic and cadmium toxicity. Altern Med Rev, 2003, 8(2): 106-128
74. Perry J, Carol A. Role of a Candida albicans P12Type ATPase in resistance to copper and silver ion toxicity. JBacteriol, 2000, 182 (17): 4899-4905
75. Pfeiffer J, Guhl J, et al. Magnesium uptake by CorA is essential for viability of the gastric pathogen Helicobacter pylori. Infect Immun, 2002, 70(7): 3930-3934
76. Remacle J, Mugaruza I, Fransolet M. Cadmium removal a strain of Alcaligenes denitrificam isolated from a metal-polluted pond. Water Resourses, 1992, 26(7): 923-926
77. Rensing C, Pribyl T, Nies D H. New Functions for the three Subunits of the CzcCBA Cation-Proton-Antiporter. J Bacteriol, 1997, 179(22): 6871-6879
78. Riggle P J, Kumamoto C A. Rolo of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity. J Bacteriol, 2000, 182(17): 4899-4905
79. Rouch D A, Lee B T D, Morby A P. Understanding cellular response to toxic agents: a model for mechanism choice in bacterial metal resistance. J Ind Microbiol, 1995, 14:132-141
80. Scott S, Veronica W, et al. Cloning and expression of CadD , a new cadmium resistance gene of Staphylococcus aureus. J Bacteriol, 1999, 181(13): 4071-4075
81. Sermon J, Wevers E M, et al. CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress. Appl Environ Microbiol, 2005, 71(11): 6515-6523
82. Sharma P, Balkwill D L, Frenkel A, et al. A new Klebsiella plantcola strain(Cd)) grows anaerobically at high cadmium concentrations and precipitates cadmium sulfide. Appl Environ Microbiol, 2000, 66(7): 3083-3087
83. Silver S, Phung L T. Bacterial heavy metal resistance:New surprises. Annu Rev Microbiol, 1996, 50: 753-789.
84. Silver S, Walderhaug M. Gene regulation and chromosome determined inorganic ion transport in bacteria. Microbiol Rev, 1992, 56: 195-228
85. Silver S. Bacterial Resistance to Toxic Metal Ions-a Review. Gene, 1996, 179: 9-19
86. Smith K, Novick R P. Genetic studies on plasmid-linked cadmium resistance in Staphylococcus aureus. J Bacteriol, 1972, 112: 761-772
87. Smith R L, Banks J L, et al. Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coll. Identification of a new class of transport protein. J Biol Chem, 1993, 268(19): 14071-14080
88. Smith R L, Gottlieb E, Kucharski L M, Maguire M E. Functional similarity between archaeal and bacterial CorA magnesium transporters. J Bacteriol, 1998, 180(10): 2788-2791
89. Smith R L, Maguire M E. Distribution of the CorA Mg2+ transport system in gram-negative bacteria. J Bacteriol, 1995, 177(6): 1638-1640
90. Tanja K, Ralph J, et al. Silver based crystalline nanoparticles, microbially fabricated. Appl Phys Sci/Microbiol, 1999,96(24): 13611-1361
91. Theocharis S E, Margeli A P, et al. Metallothionein: a multifunctional protein from toxicity to cancer. Int J Biol Markers, 2003,18(3): 162-169
92. Timmis K N. Pseudomonas putida: a cosmopolitan opportunist par excellence. Environ Microbiol, 2002, 4: 779-781
93. Tripathi M, Munot H P, Shouche Y, Meyer J M, Goel R. Isolation and functional characterization of siderophore-producing lead- and cadmium-resistant Pseudomonas putida KNP9. Curr Microbiol, 2005, 50: 233-237
94. Vall M, Lorenzo V, Atrian S, et al. Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption. J Inorg Biochem, 2000, 79(1-4): 219-223
95. van der Lelie D, Schwuchow T, Schwidetzky U. Two component regulatory system involved in transcriptional control of heavy metal homoeostasis in Alcaligenes eutrophus. Mol Microbiol, 1997, 23: 493-503
96. Vasak M. Advances in metallothionein structure and functions. J Trace Elem Med Biol, 2005, 19(1): 13-17
97. Wang C L, Michels P C, Dawson S C, et al. Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture. Appl Environ Microbiol, 1997, 63(10): 4075-4078.
98. Wang S Z, Chen Y, et al. Escherichia coli CorA periplasmic domain functions as a homotetramer to bind substrate. J Biol Chem, 2006, 281(37): 26813-26820
2． 陈玉成．污染环境生物修复工程．北京：化学工业出版社，2003
3． 胡南，赵斌．一株耐镉恶臭假单胞菌的分离鉴定及其重要的镉抗性相关基因的克 隆．[硕士学位论文]．武汉：华中农业大学，2006
4． 贾广宁．重金属污染的危害与防治．有色矿冶，2004，20(1)：39-42
5．李洪亮，陈玉成．污水处理中重金属的毒理学研究进展．微量元素与健康研究， 2006，23(1)：49-53
6． 田建民．用微生物外红硫螺菌属形成的生物聚合物去除废水中的重金属．太原 理工大学学报，1999，30(2)：175-178
7． 王保军，杨惠芳．微生物与重金属的相互作用．重庆环境科学，1996，18(1)： 35-38
8． 卫扬保．微生物生理学．高等教育出版社，1989
9． 谢曙光．重金属对生物除磷影响的研究．[硕士学位论文]．西安理工大学，2000
10．叶锦韶，尹华，彭辉．微生物抗重金属毒性研究进展．环境污染治理技术与设备， 2002，3(4)：1-4
11.Alonso A, Sanchez P, et al. Stenotrophomonas maltophilia D457R contains a cluster of genes from gram-positive bacteria involved in antibiotic and heavy metal resistance. Antimicrob Agents Chemother, 2000, 44(7): 1778-1782
12. Anton A, Grosse C, et al. CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34. J Bacteriol, 1999, 181(22): 6876-6881
13. Banjerdkij P, Vattanaviboon P, Mongkolsuk S. Exposure to cadmium elevates expression of genes in the OxyR and OhrR regulons and induces cross-resistance to peroxide killing treatment in Xanthomonas campestris. Appl Environ Microbiol, 2005, 71: 1843-1849
14. Berson O, Lidstrom M E. Cloning and characterization of corA, a gene encoding a copper-repressible polypeptide in the type I methanotroph, Methylomicrobium albus BG8. FEMS Microbiol Lett, 1997, 148(2): 169-174
15. Bertini I, Hartmann H J, Klein T, et al. High resolution solution structure of the protein part of Cu7 metallothionein. Eur J Biochem, 2000, 267 (4): 1008-1018
16. Blattner F R, Plunkett G R, Bloch C A. The complete genome sequence of Escherichia coli K-12. Science, 1997, 277: 1453-1474
17. Bloss T, Clemens S, et al. Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes. Planta, 2002, 214(5): 783-791
18. Borremans B, Hobman J L, et al. Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Barteriol, 2001, 183 (19): 5651-5658
19. Caccavo F J. Protein-mediated adhesion of the dissimilatory Fe( III )-reducing bacterium Shewanella alga BrY to hydrous ferric oxide. Appl Environ Microbiol, 1999,65:5017-5022.
20. Canovas D, Cases I, de Lorenzo V. Heavy metal tolerance and metal homeostasis in Pseudomonas putida as revealed by complete genome analysis. Environ Microbiol, 2003, 5: 1242-1256
21. Claudia G, Mark K, et al. Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem, 2000, 275(41): 32310-32316
22. Coyle P, Philcox J C, et al. Metallothionein: the multipurpose protein. Cell Mol Life Sci, 2002, 59(4): 627-47
23. Dey S, Papadopoulou B, Haimeur A, Roy G, Grondin K, Dou D, Rosen B P, Ouellette M. High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system. Mol Biochem Parasitol, 1994, 67: 49-57
24. Dey S, Rosen B P. Dual mode of energy coupling by the oxyanion-translocating ArsB protein. J Bacteriol, 1995, 1770: 385-389
25. Dopson M, Baker-Austin C, Koppineedi P R, Bond P L. Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic micro-organisms. Microbiology, 2003, 149(8): 1959-1970
26. Dopson M, Lindstrom E B. Potential role of thiobacillus caldus in arsenopyrite bioleaching. Appl Environ Microbiol, 1999, 65(11): 36-40
27. Eccles H. Treatment of metal-contaminated wastes: why select a biological process? Trends Biotechnol, 1999, 17(12): 462-465
28. Endo G, Silver S. CadC, the transcriptional regulatory protein of the cadmium resistance system of S taphylococcus aureus plasmid pI258. J Bacteriol, 1995, 177(15): 4437-4441
29. Fagan M J, Saier M H J. P-typ ATPases of eukaryotes and bacteria: sequence comparisons and construction of phylogenetic trees. J Mol Evol, 1994, 38: 57-99
30. Gadd G M. Bioremedial potential of microbiol mechanisms of metal mobilization and immobilization. Curr Opin Biotechnol, 2000, 11: 271-279.
31. Gonzalez-Guerrero M, Azcon-Aguilar C, et al. Characterization of a Glomus intraradices gene encoding a putative Zn transporter of the cation diffusion facilitator family. Fungal Genet Biol, 2005, 42(2): 130-40.
32. Grosse C, Anton A, et al. Identification of a regulatory pathway that controls the heavy-metal resistance system Czc via promoter czcNp in Ralstonia metallidurans. Arch Microbiol, 2004, 182(2-3): 109-118
33. Gross C, Kelleher M, Iyer V R, et al. Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem, 2000, 275(41): 32310-32316
34. Hao Z, Chen S, Wilson D B. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum. Appl Environ M icrobiol, 1999, 65(11): 4746-4752
35. Haney C J, Grass G, et al. New developments in the understanding of the cation diffusion facilitator family. J Ind Microbiol Biotechnol, 2005, 32(6): 215-226
36. Hassan M T, van der Lelie D, Springael D, Romling U, Ahmed N, Mergeay M. Identification of a gene cluster, czr, involved in cadmium and zinc resistance in pseudomonas aeruginosa. Gene, 1999, 238: 417-425
37. Heaton A C, Rugh C L, Kim T, et al. Toward detoxifying mercury-polluted aquatic sediments with rice genetically engineered for mercury resistance. Environ Toxicol Chem, 2003, 22(12): 2940-2947
38. Hernandez A, Mellado R P, Martinez J L. Metal accumulation and vanadium -induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae. Appl Environ Microbiol, 1998, 64(11): 4317-4320
39. Horitsu H, Yamamoto K, Wachi S, Kawai K, Fukuchi A. Plasmid-determined cadmium resistance in Pseudomonas putida GAM-1 isolated from soil. J Bacteriol, 1986,165:334-335
40. Hu N, Zhao B. Key genes involved in heavy-metal resistance in Pseudomonas putida CD2. FEMS Microbiol Lett, 2007, 267: 17-22
41. Inoue Y, Kobayashi S, Kimura A. Cloning and phenotypic characterization of a gene enhancing resistance against oxidative stress in Saccharomyces cerevisiae. J Ferment Bioeng, 1993, 75:327-331
42. Jin Y H, Clark A B, Slebos R J, Al-Refai H, Taylor J A, Kunkel T A, Resnick M A, Gordenin D A. Cadmium is a mutagen that acts by inhibiting mismatch repair. Nat Genet, 2003, 34: 326-329
43. Jonathan J, Alice L, et al. Cd( II)-responsive and constitutive mutants implicate a novel domain in MerR. J Bacteriol, 1999, 181(11): 3462-3471
44. Kagi J H. Overview of metallothionein. Methods Enzymol, 1991, 205: 613-626
45. Kambe T, Suzuki T, et al. Sequence similarity and functional relationship among eukaryotic ZIP and CDF transporters. Genomics Proteomics Bioinformatics, 2006, 4(1): 1-9
46. Kannan G M, Tripathi N, et al. Toxic effects of arsenic (III) on some hematopoietic and central nervous system variables in rats and guinea pigs. J Toxicol Clin Toxicol, 2001, 39(7): 675-682
47. Khunajakr N, Liu C Q, Charoenchai P. A plasmid-encoded two-component regulatory system involved in copper-inducible transcription in Lactococcus lactis. Gene, 1999, 229: 229-235
48. Kim D, Gustin J L, et al. The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae. Plant J, 2004, 39(2): 237-251
49. Kurdihaidar B, Horn D K, Flittner D E, Heath D, Fink L, Naredi P, Howell S B. Dual cytoplasmic and nuclear distribution of the novel arsenite-stimulated human ATPase (hASNA-I). J Cell Biochem, 1998,71: 1-10
50. Kuyucak N, Volesky B. Biosorbents for recovery of metals from industrial solutions. Biotechnol Let, 1988, 10(2): 137-142
51. Lee S W, Glickmann E, Cooksey D A. Chromosomal locus for cadmium resistance in Pseudomonas putida consisting of a cadmium-transporting ATPase and a MerR family response regulator. Appl Environ Microbiol, 2001, 67: 1437-1444
52. Legatzki A, Grass G, Anton A, Rensing C, Nies D H. Interplay of the Czc system and two P-type ATPases in conferring metal resistance to Ralstonia metallidurans. J Bacteriol, 2003, 185: 4354-4361
53. Legatzki A, Franke S, et al. First step towards a quantitative model describing Czc-mediated heavy metal resistance in Ralstonia metallidurans. Biodegradation, 2003, 14(2): 153-168
54. Leedjarv A, Ivask A, Virta M. The interplay of different transporters in the mediation of divalent heavy metal resistance in Pseudomonas putida KT2440. J Bacteriol, 2007, doi:10.1128/JB.01494-07
55. Liu J, Gladysheva T B, Lee L, Rosen B P. Identification of an essential cysteinyl residue in the ArsC arsenate reductase of plasmid 8773. Biochemistry, 1995, 34: 13472-13476
56. Liu J, Rosen B P. Ligand interactions of the ArsC arsenate reductase. J Biol Chem, 1997,272:21084-21089
57. Lloyd J R. Microbial reduction of metals and radionuclides. FEMS Microbiol Rev, 2003, 27(2-3): 411-425
58. Lunin V V, Dobrovetsky E, et al. Crystal structure of the CorA Mg2+ transporter. Nature, 2006, 440(7085): 833-837
59. Maguire M E. The structure of CorA: a Mg2+-selective channel. Curr Opin Struct Biol, 2006, 16(4): 432-438
60. Mergeay M, Nies D, et al. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol, 1985, 162(1): 328-334
61. Mejare M, Bulow L. Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends Biotechnol, 2001, 19(2): 67-73
62. Miyabe S, Izawa S, et al. Expression of ZRC1 coding for suppressor of zinc toxicity is induced by zinc-starvation stress in Zap1-dependent fashion in Saccharomyces cerevisiae. Biochem Biophys Res Commun, 2000, 276(3): 879-884
63. Misra T K.Bacterial resistance to inorganic mercucy salts and organomercurials. Plasmid, 1992,27:4-16
64. Moncrief M B, Maguire M E. Magnesium transport in prokaryotes. J Biol Inorg Chem, 1999, 4(5): 523-527
65. Navarre C, Wu L F, Mandrand B M A. The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system for nickel. Mol Microbiol, 1993,9:1181-1191
66. Nelson D L, Kennedy E P. Magnesium transport in Escherichia coli. Inhibition by cobaltous ion. J Biol Chem, 1971, 246(9): 3042-3049
67. Nies D H. CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus. J Bacteriol, 1992, 174(24): 8102-8110
68. Nies D H. Microbiol heavy-metal resistance. Appl Microbiol Biotechnol, 1999, 51(6): 730-750
69. Nies D H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev, 2003, 27: 313-339
70. Nies D H, Silver S. Ion efflux systems involved in bacterial metal resistances. J Ind Microbiol, 1995, 14(2): 186-199
71. Pardo R, Herguedas M, Barrado E, Vega M. Biosorption of cadmium, copper, lead and zinc by inactive biomass of Pseudomonas Putida. Anal Bioanal Chem, 2003, 376: 26-32
72. Park M H, Wong B B, Lusk J E. Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology. J Bacteriol, 1976, 126(3): 1096-1103
73. Patrick L. Toxic metals and antioxidants: Part II. The role of antioxidants in arsenic and cadmium toxicity. Altern Med Rev, 2003, 8(2): 106-128
74. Perry J, Carol A. Role of a Candida albicans P12Type ATPase in resistance to copper and silver ion toxicity. JBacteriol, 2000, 182 (17): 4899-4905
75. Pfeiffer J, Guhl J, et al. Magnesium uptake by CorA is essential for viability of the gastric pathogen Helicobacter pylori. Infect Immun, 2002, 70(7): 3930-3934
76. Remacle J, Mugaruza I, Fransolet M. Cadmium removal a strain of Alcaligenes denitrificam isolated from a metal-polluted pond. Water Resourses, 1992, 26(7): 923-926
77. Rensing C, Pribyl T, Nies D H. New Functions for the three Subunits of the CzcCBA Cation-Proton-Antiporter. J Bacteriol, 1997, 179(22): 6871-6879
78. Riggle P J, Kumamoto C A. Rolo of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity. J Bacteriol, 2000, 182(17): 4899-4905
79. Rouch D A, Lee B T D, Morby A P. Understanding cellular response to toxic agents: a model for mechanism choice in bacterial metal resistance. J Ind Microbiol, 1995, 14:132-141
80. Scott S, Veronica W, et al. Cloning and expression of CadD , a new cadmium resistance gene of Staphylococcus aureus. J Bacteriol, 1999, 181(13): 4071-4075
81. Sermon J, Wevers E M, et al. CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress. Appl Environ Microbiol, 2005, 71(11): 6515-6523
82. Sharma P, Balkwill D L, Frenkel A, et al. A new Klebsiella plantcola strain(Cd)) grows anaerobically at high cadmium concentrations and precipitates cadmium sulfide. Appl Environ Microbiol, 2000, 66(7): 3083-3087
83. Silver S, Phung L T. Bacterial heavy metal resistance:New surprises. Annu Rev Microbiol, 1996, 50: 753-789.
84. Silver S, Walderhaug M. Gene regulation and chromosome determined inorganic ion transport in bacteria. Microbiol Rev, 1992, 56: 195-228
85. Silver S. Bacterial Resistance to Toxic Metal Ions-a Review. Gene, 1996, 179: 9-19
86. Smith K, Novick R P. Genetic studies on plasmid-linked cadmium resistance in Staphylococcus aureus. J Bacteriol, 1972, 112: 761-772
87. Smith R L, Banks J L, et al. Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coll. Identification of a new class of transport protein. J Biol Chem, 1993, 268(19): 14071-14080
88. Smith R L, Gottlieb E, Kucharski L M, Maguire M E. Functional similarity between archaeal and bacterial CorA magnesium transporters. J Bacteriol, 1998, 180(10): 2788-2791
89. Smith R L, Maguire M E. Distribution of the CorA Mg2+ transport system in gram-negative bacteria. J Bacteriol, 1995, 177(6): 1638-1640
90. Tanja K, Ralph J, et al. Silver based crystalline nanoparticles, microbially fabricated. Appl Phys Sci/Microbiol, 1999,96(24): 13611-1361
91. Theocharis S E, Margeli A P, et al. Metallothionein: a multifunctional protein from toxicity to cancer. Int J Biol Markers, 2003,18(3): 162-169
92. Timmis K N. Pseudomonas putida: a cosmopolitan opportunist par excellence. Environ Microbiol, 2002, 4: 779-781
93. Tripathi M, Munot H P, Shouche Y, Meyer J M, Goel R. Isolation and functional characterization of siderophore-producing lead- and cadmium-resistant Pseudomonas putida KNP9. Curr Microbiol, 2005, 50: 233-237
94. Vall M, Lorenzo V, Atrian S, et al. Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption. J Inorg Biochem, 2000, 79(1-4): 219-223
95. van der Lelie D, Schwuchow T, Schwidetzky U. Two component regulatory system involved in transcriptional control of heavy metal homoeostasis in Alcaligenes eutrophus. Mol Microbiol, 1997, 23: 493-503
96. Vasak M. Advances in metallothionein structure and functions. J Trace Elem Med Biol, 2005, 19(1): 13-17
97. Wang C L, Michels P C, Dawson S C, et al. Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture. Appl Environ Microbiol, 1997, 63(10): 4075-4078.
98. Wang S Z, Chen Y, et al. Escherichia coli CorA periplasmic domain functions as a homotetramer to bind substrate. J Biol Chem, 2006, 281(37): 26813-26820