Molecular Mechanism of Resistance of heavy Metal Resistant Bacteria and Their Application of to Heavy Metal-Containing Waste Water.

重金属耐药菌的耐药分子机制及其在含重金属废水中的应用。

• 批准号: 02660111
• 负责人: KAWAI Keiichi
• 金额: $ 1.15万
• 依托单位: Gifu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02660111/
• 关键词: Pseudomonas putida GAM-1; Heavy Metal-Resistant Gene; Heavy Metal-Resistant Plasmid; Heavy Metal-Resistant E. coli transformants; Pseudomonas ambigua G-1; Cr (VI) Reductase; Heavy Metal-Containing Water; Bacterial Cr (VI) Reduction; カドミウム耐性細菌; 六価

l. MOLECULAR CLONING OF HEAVY METAL-RESISTANCE GENE.(1) P. Putida GAM-1 harboring pGUIOO can grow at 7mM CdCl_2. The Cd(II)-resistance was encoded in pGUlOO. By shot-gun cloning, a Cd(II)-resistant Ecoli C600 transformant was obtained, and it harbored a plasmid (pCDR1, 5.0 kb) which was constructed by BamHI treatment. But, BamHI could cut only one site in pCDR1. Southern analysis was done by using BamHI-EcoRI DNA fragment of pCDR1. One of BamHI-digested pGU1OO fragments was hybridized with BamHI-EcoRI fragment. DNA sequence of the BamHI-EcoRI fragment was highly homologous for that of IS1. (2) P. ambigua G-1 harboring 7 plasmids can grow at 1OOmM K_2CrO_4. Ecoli C600 was transformed to be Cr(VI)-resistant with the plasmids. Three Cr(VI)-resistant Ecoli C600 transformants were obtained, and they harbored 7.4 kb plasmid in common.2. MECHANISM OF HEAVY METAL-RESISTANCE.(1) Cd(II)-resistance of P. putida GAM-1 was induced by 1mM CdCl_2 , but that of E. coli C600(pGUIOO) was constitutive. T … More he Cd(II) content of E. coli C600 was 3-fold, as compared with that of E. coli C600(pGUIOO). This reduced acccumulation of Cd(II) by this transformant was due to an energy-dependent Cd(II) efflux system. (2) Cr(VI) reductase of P. ambigua G-1 was highly purified with several columnchromatographies. The molecular weight of the enzyme was estimated to be 65, 000 and that of subunit was 25, 000, indicating that the reductase consists two identical subunits. The enzyme required NAD(P) H as electron donor. The optimal temperature and pH were 60ﾟC and 8.1-8.6, respectively. Stoichiometry of Cr(VI) reduction and ESR spectra revealed that the Cr(VI) reductase reducod Cr(VI) to Cr(VI) to Cr(III) through Cr(V) as an intermed. iate.3. MICROBIAL TREATMENT OF HEAVY METAL-CONTAINING WASTE WATER.(1) P. Putida GAM-1 and Cd(II)-resistant E. coli C600 transformants were able to grow in the presence of 3.5mM CdCl_2. As P. putida GAM-1 can not grow at below pH 5, heavy metal-containing waste water in low pH requires the neutralization of the waste water. (2) P. ambigua G-1 was capable of growing in L-broth containing 150 ppm Cr(VI) and reduced actively Cr(VI) to Cr(III). Less

L.重金属抗性基因的分子克隆(1)含有pGU 100的P. Putida GAM-1在7 mM CdCl_2下可以生长。在pGUlOO中编码Cd（II）抗性。通过鸟枪法克隆，获得了一株抗Cd（II）的大肠杆菌C600菌株，该菌株含有经BamHI酶切构建的质粒pCDR 1（5.0kb）。但BamHI只能切割pCDR 1的一个位点。用pCDR 1的BamHI-EcoRI DNA片段进行Southern分析。用BamHI-EcoR Ⅰ酶切的pGU 100片段与BamHI-EcoR Ⅰ片段杂交。BamHI-EcoRI片段的DNA序列与IS 1的DNA序列高度同源。(2)含7个质粒的歧义假单胞菌G-1能在100mMK_2CrO_4中生长。用该质粒转化大肠杆菌C600使其具有Cr（VI）抗性。获得了3株抗Cr（VI）的Ecoli C600转化子，它们均携带7.4kb质粒.抗重金属机理(1)恶臭假单胞菌GAM-1对Cd（II）的抗性可被1 mM CdCl_2诱导，而大肠杆菌GAM-1对Cd（II）的抗性可被1 mM CdCl_2诱导。coliC 600（pGU 100）为组成型。不 ...更多信息 Cd（II）含量。coliC 600与E. coliC600（pGU100）。这减少accumulation的镉（II），这是由于能量依赖的镉（II）外排系统。(2)采用多种柱层析方法对青虫G-1的Cr（VI）还原酶进行了高纯度纯化。酶的分子量为65，000，亚基分子量为25，000，表明该还原酶由两个相同的亚基组成。该酶需要NAD（P）H作为电子供体。最适温度为60 ℃，最适pH为8.1-8.6。Cr（VI）还原的化学计量学和ESR谱表明，Cr（VI）还原酶通过Cr（V）作为中间体将Cr（VI）还原为Cr（VI）再还原为Cr（III）。3.重金属废水的微生物处理(1)P. Putida GAM-1和抗Cd（II）E. coliC 600转化子能在3.5mMCdCl_2存在下生长。由于恶臭假单胞菌GAM-1不能在低于pH 5的条件下生长，低pH的含重金属废水需要对废水进行中和。(2)P. ambigua G-1能够在含有150 ppm Cr（VI）的L-肉汤中生长，并将Cr（VI）主动还原为Cr（III）。少

项目成果

Keiichi Kawai: "Expression of PlasmidーEncoded Gene for Cadmium Resistance of Pseudomonas putida GAMー1 in Escherichia coli" Journal of Fermentation and Bioengineering. 73. (1992)

Keiichi Kawai：“大肠杆菌中恶臭假单胞菌 GAM-1 镉抗性的质粒编码基因的表达”发酵与生物工程杂志 73。（1992）

Tohru SUZUKI, Naoyuki MIYATA, Hiroyuki HORITSU, Keiichi KAWAI, and Kazuhiro TAKAMIZAWA: "Catalytic Mechanism of NAD (P) H Dependent Chromate Reductase of Pseudomonas ambigua G-1 : Cr (V) Intermediate is formed during the Reducation of Cr (VI) to Cr (III).

Tohru SUZUKI、Naoyuki MIYATA、Hiroyuki HORITSU、Keiichi KAWAI 和 Kazuhiro TAKAMIZAWA：“Pseudomonas ambigua G-1 的 NAD (P) H 依赖性铬酸还原酶的催化机制：Cr (V) 中间体是在 Cr (VI) 还原过程中形成的

Tohru SUZUKI, Naoyuki MIYATA, Yuko ICHIYANAGI, Hiroyuki HORITSU, and Kazuhiro TAKAMIZAWA: "Purification and Characterization of Cr (VI) Reductase of Pseudomonas ambigua G-1." J. Bacteriol.,. (1992)

Tohru SUZUKI、Naoyuki MIYATA、Yuko ICHIYANAGI、Hiroyuki HORITSU 和 Kazuhiro TAKAMIZAWA：“Pseudomonas ambigua G-1 的 Cr (VI) 还原酶的纯化和表征。”

Tohru Suzuki: "Catalytic Mechanism of NAD(P)H Dependent Chromium (VI) Reductase of Pseduomonas ambigua G-1:Cr(V)Intermediate is formed during the Reduction of Cr(VI)to Cr(III)" Journal of Bacteriology.

Tohru Suzuki：“NAD(P)H 依赖性假单胞菌 G-1 铬 (VI) 还原酶的催化机制：Cr(V)中间体在 Cr(VI) 还原为 Cr(III) 过程中形成”细菌学杂志。

Tohru Suzuki: "Purification and Characterization of Cr(VI) Reductase of Pseudomonas ambigua G-1" Journal of Bacteriology.

Tohru Suzuki：“Pseudomonas ambigua G-1 的 Cr(VI) 还原酶的纯化和表征”细菌学杂志。