α-酮戊二酸脱氢酶基因在嗜酸性氧化硫硫杆菌中的表达研究
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摘要
嗜酸性氧化硫硫杆菌广泛用于生物冶金、煤的脱硫和含硫废水及垃圾的处理。酶学分析表明,嗜酸性氧化硫硫杆菌缺乏TCA循环中的关键酶α-酮戊二酸脱氢酶等,不能单纯通过TCA循环氧化碳水化合物获得能量,其不完全的TCA循环仅提供中间产物参与合成细胞的部分碳架。这类细菌的自养生活方式决定了这些细菌生长缓慢、代时长、细胞得率低。这不仅限制了嗜酸性氧化硫硫杆菌在工业上的应用,也给生化检测、蛋白质和酶的分离及DNA的提取等带来困难。要从根本上解决这一问题需要用遗传学的方法对菌种进行改良,使其生长代谢适合于实际应用的要求。
本文的目的是将克隆的异养性大肠杆菌α-酮戊二酸脱氢酶复合体基因sucAB与lpdA导入专性自养极端嗜酸性氧化硫硫杆菌中表达,获得可以表达α-酮戊二酸脱氢酶活性,并能够利用有机质快速生长的嗜酸性氧化硫硫杆菌工程菌,并进一步研究有机质对基因工程菌生长的影响机制以及工程菌对有机质的利用情况等。为改造这类细菌并应用于工农业生产提供理论和方法。
实验室前期已经以E.coli K12菌株基因组DNA作为模板,利用PCR方法扩增得到大肠杆菌α-酮戊二酸脱氢酶复合体sucAB与lpdA全基因序列,并连接到质粒pUC18载体中。在此基础上,本文进行了如下研究:
把大肠杆菌α-酮戊二酸脱氢酶复合体sucAB与lpdA基因从pUC18上亚克隆到具有广泛寄主范围的质粒pJRD215上,构建了含大肠杆菌α-酮戊二酸脱氢酶基因的重组质粒pJRD215-sucAB-lpdA。结果表明,重组质粒pJRD215-sucAB-lpdA所携带的sucAB与lpdA基因基因能够在大肠杆菌中表达出具有活性的酶蛋白,而且保留了载体上原有的Km和Sm抗性。
将重组质粒pJRD215-sucAB-lpdA引入整合有tra基因的E.coli SM10中,以E.coliSM10(pJRD215-sucAB-lpdA)作为供体菌,野生型Acidithiobacillus thiooxidansATCC19377作为受体菌在滤膜上进行接合转移。质粒pJRD215-sucAB-lpdA在tra基因的作用下以较高的频率(2.4×10~(-6))从大肠杆菌转移到了A.thiooxidans ATCC19377菌株中,并通过质粒提取及菌落PCR进行了验证。酶活性测定表明,在对照野生型菌株的细胞提取液中未检测到α-酮戊二酸脱氢酶活性,而含有重组质粒的嗜酸性氧化硫硫杆菌细胞提取液中可检测到。这表明,大肠杆菌的启动子可以被嗜酸性氧化硫硫杆菌的RNA聚合酶识别并启动转录,α-酮戊二酸脱氢酶复合体基因能够表达出具有活性的酶,因而推测极端嗜酸性硫杆菌在基因表达机制方面与异养性大肠杆菌可能存在相似之处。利用RT-PCR的方法从转录水平上检测基因的转录,证明野生型嗜酸性氧化硫硫杆菌存在代谢缺陷,而重组菌中可以检测到sucAB与lpdA基因mRNA的转录。但是无论从蛋白水平还是酶活水平上检测,重组菌中表达的α-酮戊二酸脱氢酶活性非常低,说明在嗜酸性氧化硫硫杆菌和大肠杆菌之间在基因表达和调控方面还存在一定差异。质粒的稳定性测定表明,在无选择压力条件下连续传代50次,质粒在嗜酸性氧化硫硫杆菌的保存率仍可达到71%左右,说明质粒在嗜酸性氧化硫硫杆菌中比较稳定。
研究了氧化硫硫杆菌基因工程菌在Starkey-S~0液体培养基中添加不同浓度的葡萄糖、有机酸(α-酮戊二酸、柠檬酸和丙酮酸)的生长状况。结果表明,工程菌可以更有效的利用葡萄糖。而α-酮戊二酸、柠檬酸对野生型与卫程菌的生长没有明显影响,这可能与细胞的选择透性有关。研究还发现工程菌可以解除丙酮酸的抑制作用,说明中间代谢产物的积累对细胞确实有毒害作用。虽然葡萄糖对嗜酸性氧化硫硫杆菌的生长有促进作用,但在不提供硫作为能源时无法生长,说明硫杆菌不具备利用有机物产生ATP的电子传递系统,因而对有机物的利用有限。
本文首次将大肠杆菌α-酮戊二酸脱氢酶复合体基因引入嗜酸性氧化硫硫杆菌中,sucAB与lpdA基因在自身启动子的控制下在宿主菌中获得了表达,证明了异养菌中心代谢途径的酶复合体基因(α-酮戊二酸脱氢酶复合体)能够在专性自养极端嗜酸性氧化硫硫杆菌中表达装配。尽管表达的酶活性较低,所构建的工程菌能够更加有效地利用葡萄糖并消除丙酮酸等中间代谢物的抑制作用,为解决实际应用中菌体生长过慢,效率低的难题提供了一条可能的途径。本研究不仅可以从理论上为揭示细菌自养与异养的关系提供实验依据,而且在应用方面可为构建能够利用有机质快速生长的基因工程菌提供实验指导。
Acidithiobacillus thiooxidans is a gram-negative, extremely acidophilic obligately autotrophic bacterium, which can obtain energy from the chemolithotrophic oxidation of inorganic sulphur and its compounds and use this energy to support autotrophic growth on carbon dioxide. It has been used widely in metal leaching from mineral ores, in the desulfurization of coal, and in the treatment of sulfur containing waste water and garbage. However, the slow growth rate and the low cell yield of this organism and its sensitivity to heavy metals have limited its further use. Enzymological research revealed that A. thiooxidans is deficient in some key enzymes of the EMP pathway and Krebs cycle, such as phosphofructokinase, andα-ketoglutarate dehydrogenase. So, these organisms could not respire organic substance adequately and obtain energy from them.
α-ketoglutarate dehydrogenase complex is an important enzyme of the Krebs cycle. The purpose of this study is to introduce the cloned sucAB and lpdA genes for this enzyme into A. thiooxidans and to construct new A. thiooxidans gene engineering strain with the expressedα-ketoglutarate dehydrogenase activities and better growth rates on account of metabolism of organic compounds.
In the former study, the two sucAB and lpdA gene fragments coding forα-ketoglutarate dehydrogenase complex, which contain its own promoter and ribosome binding site, had been amplified separately by the method of PCR using the genomic DNA of E. coli K12 as the template, and cloned together into plasmid pUC18. In this study, the sucAB and lpdA genes were first subcloned into the wide-host-range plasmid pJRD215 to construct the recombinant plasmid pJRD215-sucAB-lpdA. The agarose gel electrophoretic pattern of recombinant plasmid pJRD215-sucAB-lpdA digested by EcoR I and Hind III showed that pJRD215-sucAB-lpdA carried the heterogeneous 4.8 kb sucAB and 1.8 kb lpdA gene fragments. The specific active ofα-ketoglutarate dehydrogenase expressed by the sucAB and lpdA genes in recombinant plasmid pJRD215-sucAB-lpdA could be detected and the corresponding proteins were verified by the SDS-PAGE. By the way, both the genes for Km~r and Sm~r in pJRD215-sucAB-lpdA could be expressed in E. coli cells.
The recombinant plasmid pJRD215- sucAB-lpdA was then transformed into E. coli SM10. Using E. coli SM10 (pJRD215-sucAB-lpdA) as the donors and wild type A. thiooxidans ATCC19377 as the recipients, the recombinant plasmid pJRD215-sucAB-lpdA could be mobilized into A. thiooxidans strains with the aid of tra gene on the genomic DNA of E. coli SM10. The transfer frequency of plasmid pJRD215-sucAB-lpdA from E. coli SM10 to A. thiooxidans was 2.4×10~(-6) using Km as the selective marker. The presence of pJRD215-sucAB-lpdA in A. thiooxidans transconjugants were verified by plasmid isolation and transconjugants colony PCR.
The specific activity ofα-ketoglutarate dehydrogenase was not detectable in the wild type A. thiooxidans ATCC19377, which revealed the natural deficient of this enzyme in A. thiooxidans strains. However, it was detectable in the gene engineering A. thiooxidans ATCC19377 harboring plasmid pJRD215- sucAB-lpdA, although at a very low level. The expression of sucAB and lpdA genes in A. thiooxidans might suggest that the promoter sequences of E. coli could be correctly recognized by the RNA polymerase of autotrophic bacterium. And these studies also implied that the gene expression system might be similarin E. coli and A. thiooxidans, though there were great physiological differences between them.
The expression of sucAB and lpdA genes in A. thiooxidans ATCC19377 (pJRD215-sucAB-lpdA) was also detected at the level of transcription and translation. Agarose gel electrophoresis of RT-PCR products confirmed the transcriptions of sucB and lpdA genes. However, the enzyme activities measured in A. thiooxidans ATCC19377 (pJRD215-sucAB-lpdA) were unexpectedly low whether at the protein level or at the enzymatic activity level. This might indicate that there are some differences in the gene expression and control system between A. thiooxidans and E. coli. The stability of plasmid pJRD215-sucAB-lpdA in A. thiooxidans ATCC19377 was determined by checking for km and Sm resistance. About 71% of A. thiooxidans cells carried the recombinant plasmids after being cultured for 5 generations without selective pressure, which showed that pJRD215-sucAB-lpdA was maintained consistently in A thiooxidans.
The growth of A. thiooxidans ATCC19377 (pJRD215- sucAB-lpdA) was also studied by adding different concentration of glucose, organic acids (α-ketoglutarate acid, citrate acid and pyruvate acid) in Starkey-S~0 liquid medium. Glucose caused some stimulation on the cell growth of gene engineering strain, butα-ketoglutarate acid and citrate acid showed no effect on the growth of both the wild type and gene engineering strains under the studied concentration level. Pyruvate acid could inhibit the growth of both the wild type and gene engineering strains, but the inhibition could be partially relieved by the gene engineering strains. Although glucose could facilitate the cell growth of A. thiooxidans, but only under the supply of inorganic sulfur, which might indicate the absence of electron transfer system to produce ATP by the metabolism of organic compounds. So, only limited organic compounds could be metabolized by A. thiooxidans.
In conclusion, the genes forα-ketoglutarate dehydrogenase complex were introduced into A. thiooxidans for the first time, and the sucAB and lpdA genes could be expressed in A. thiooxidans under its own promoter. This indicated that the heterotrophic complicated genes for the enzymes (α-ketoglutarate dehydrogenase complex) in the central metabolic pathway could be expressed and assembled in the extremely acidophilic, obligately chemolithoautotrophic A. thiooxidans. Though possessing low enzymatic activities, the constructed gene engineering strains could metabolize glucose more efficient and mitigate the inhibition caused by the metabolic intermediates such as pyruvate acid, and the faster growth strains constructed would be more useful for the application. Our work will not only open a way to investigate the phylogenetic relationship between heterotrophic and autotrophic bateria, but also provide a new insight into how to improve the growth rate and leaching performance of the obligately autotrophic bacteria.
本文的目的是将克隆的异养性大肠杆菌α-酮戊二酸脱氢酶复合体基因sucAB与lpdA导入专性自养极端嗜酸性氧化硫硫杆菌中表达,获得可以表达α-酮戊二酸脱氢酶活性,并能够利用有机质快速生长的嗜酸性氧化硫硫杆菌工程菌,并进一步研究有机质对基因工程菌生长的影响机制以及工程菌对有机质的利用情况等。为改造这类细菌并应用于工农业生产提供理论和方法。
实验室前期已经以E.coli K12菌株基因组DNA作为模板,利用PCR方法扩增得到大肠杆菌α-酮戊二酸脱氢酶复合体sucAB与lpdA全基因序列,并连接到质粒pUC18载体中。在此基础上,本文进行了如下研究:
把大肠杆菌α-酮戊二酸脱氢酶复合体sucAB与lpdA基因从pUC18上亚克隆到具有广泛寄主范围的质粒pJRD215上,构建了含大肠杆菌α-酮戊二酸脱氢酶基因的重组质粒pJRD215-sucAB-lpdA。结果表明,重组质粒pJRD215-sucAB-lpdA所携带的sucAB与lpdA基因基因能够在大肠杆菌中表达出具有活性的酶蛋白,而且保留了载体上原有的Km和Sm抗性。
将重组质粒pJRD215-sucAB-lpdA引入整合有tra基因的E.coli SM10中,以E.coliSM10(pJRD215-sucAB-lpdA)作为供体菌,野生型Acidithiobacillus thiooxidansATCC19377作为受体菌在滤膜上进行接合转移。质粒pJRD215-sucAB-lpdA在tra基因的作用下以较高的频率(2.4×10~(-6))从大肠杆菌转移到了A.thiooxidans ATCC19377菌株中,并通过质粒提取及菌落PCR进行了验证。酶活性测定表明,在对照野生型菌株的细胞提取液中未检测到α-酮戊二酸脱氢酶活性,而含有重组质粒的嗜酸性氧化硫硫杆菌细胞提取液中可检测到。这表明,大肠杆菌的启动子可以被嗜酸性氧化硫硫杆菌的RNA聚合酶识别并启动转录,α-酮戊二酸脱氢酶复合体基因能够表达出具有活性的酶,因而推测极端嗜酸性硫杆菌在基因表达机制方面与异养性大肠杆菌可能存在相似之处。利用RT-PCR的方法从转录水平上检测基因的转录,证明野生型嗜酸性氧化硫硫杆菌存在代谢缺陷,而重组菌中可以检测到sucAB与lpdA基因mRNA的转录。但是无论从蛋白水平还是酶活水平上检测,重组菌中表达的α-酮戊二酸脱氢酶活性非常低,说明在嗜酸性氧化硫硫杆菌和大肠杆菌之间在基因表达和调控方面还存在一定差异。质粒的稳定性测定表明,在无选择压力条件下连续传代50次,质粒在嗜酸性氧化硫硫杆菌的保存率仍可达到71%左右,说明质粒在嗜酸性氧化硫硫杆菌中比较稳定。
研究了氧化硫硫杆菌基因工程菌在Starkey-S~0液体培养基中添加不同浓度的葡萄糖、有机酸(α-酮戊二酸、柠檬酸和丙酮酸)的生长状况。结果表明,工程菌可以更有效的利用葡萄糖。而α-酮戊二酸、柠檬酸对野生型与卫程菌的生长没有明显影响,这可能与细胞的选择透性有关。研究还发现工程菌可以解除丙酮酸的抑制作用,说明中间代谢产物的积累对细胞确实有毒害作用。虽然葡萄糖对嗜酸性氧化硫硫杆菌的生长有促进作用,但在不提供硫作为能源时无法生长,说明硫杆菌不具备利用有机物产生ATP的电子传递系统,因而对有机物的利用有限。
本文首次将大肠杆菌α-酮戊二酸脱氢酶复合体基因引入嗜酸性氧化硫硫杆菌中,sucAB与lpdA基因在自身启动子的控制下在宿主菌中获得了表达,证明了异养菌中心代谢途径的酶复合体基因(α-酮戊二酸脱氢酶复合体)能够在专性自养极端嗜酸性氧化硫硫杆菌中表达装配。尽管表达的酶活性较低,所构建的工程菌能够更加有效地利用葡萄糖并消除丙酮酸等中间代谢物的抑制作用,为解决实际应用中菌体生长过慢,效率低的难题提供了一条可能的途径。本研究不仅可以从理论上为揭示细菌自养与异养的关系提供实验依据,而且在应用方面可为构建能够利用有机质快速生长的基因工程菌提供实验指导。
Acidithiobacillus thiooxidans is a gram-negative, extremely acidophilic obligately autotrophic bacterium, which can obtain energy from the chemolithotrophic oxidation of inorganic sulphur and its compounds and use this energy to support autotrophic growth on carbon dioxide. It has been used widely in metal leaching from mineral ores, in the desulfurization of coal, and in the treatment of sulfur containing waste water and garbage. However, the slow growth rate and the low cell yield of this organism and its sensitivity to heavy metals have limited its further use. Enzymological research revealed that A. thiooxidans is deficient in some key enzymes of the EMP pathway and Krebs cycle, such as phosphofructokinase, andα-ketoglutarate dehydrogenase. So, these organisms could not respire organic substance adequately and obtain energy from them.
α-ketoglutarate dehydrogenase complex is an important enzyme of the Krebs cycle. The purpose of this study is to introduce the cloned sucAB and lpdA genes for this enzyme into A. thiooxidans and to construct new A. thiooxidans gene engineering strain with the expressedα-ketoglutarate dehydrogenase activities and better growth rates on account of metabolism of organic compounds.
In the former study, the two sucAB and lpdA gene fragments coding forα-ketoglutarate dehydrogenase complex, which contain its own promoter and ribosome binding site, had been amplified separately by the method of PCR using the genomic DNA of E. coli K12 as the template, and cloned together into plasmid pUC18. In this study, the sucAB and lpdA genes were first subcloned into the wide-host-range plasmid pJRD215 to construct the recombinant plasmid pJRD215-sucAB-lpdA. The agarose gel electrophoretic pattern of recombinant plasmid pJRD215-sucAB-lpdA digested by EcoR I and Hind III showed that pJRD215-sucAB-lpdA carried the heterogeneous 4.8 kb sucAB and 1.8 kb lpdA gene fragments. The specific active ofα-ketoglutarate dehydrogenase expressed by the sucAB and lpdA genes in recombinant plasmid pJRD215-sucAB-lpdA could be detected and the corresponding proteins were verified by the SDS-PAGE. By the way, both the genes for Km~r and Sm~r in pJRD215-sucAB-lpdA could be expressed in E. coli cells.
The recombinant plasmid pJRD215- sucAB-lpdA was then transformed into E. coli SM10. Using E. coli SM10 (pJRD215-sucAB-lpdA) as the donors and wild type A. thiooxidans ATCC19377 as the recipients, the recombinant plasmid pJRD215-sucAB-lpdA could be mobilized into A. thiooxidans strains with the aid of tra gene on the genomic DNA of E. coli SM10. The transfer frequency of plasmid pJRD215-sucAB-lpdA from E. coli SM10 to A. thiooxidans was 2.4×10~(-6) using Km as the selective marker. The presence of pJRD215-sucAB-lpdA in A. thiooxidans transconjugants were verified by plasmid isolation and transconjugants colony PCR.
The specific activity ofα-ketoglutarate dehydrogenase was not detectable in the wild type A. thiooxidans ATCC19377, which revealed the natural deficient of this enzyme in A. thiooxidans strains. However, it was detectable in the gene engineering A. thiooxidans ATCC19377 harboring plasmid pJRD215- sucAB-lpdA, although at a very low level. The expression of sucAB and lpdA genes in A. thiooxidans might suggest that the promoter sequences of E. coli could be correctly recognized by the RNA polymerase of autotrophic bacterium. And these studies also implied that the gene expression system might be similarin E. coli and A. thiooxidans, though there were great physiological differences between them.
The expression of sucAB and lpdA genes in A. thiooxidans ATCC19377 (pJRD215-sucAB-lpdA) was also detected at the level of transcription and translation. Agarose gel electrophoresis of RT-PCR products confirmed the transcriptions of sucB and lpdA genes. However, the enzyme activities measured in A. thiooxidans ATCC19377 (pJRD215-sucAB-lpdA) were unexpectedly low whether at the protein level or at the enzymatic activity level. This might indicate that there are some differences in the gene expression and control system between A. thiooxidans and E. coli. The stability of plasmid pJRD215-sucAB-lpdA in A. thiooxidans ATCC19377 was determined by checking for km and Sm resistance. About 71% of A. thiooxidans cells carried the recombinant plasmids after being cultured for 5 generations without selective pressure, which showed that pJRD215-sucAB-lpdA was maintained consistently in A thiooxidans.
The growth of A. thiooxidans ATCC19377 (pJRD215- sucAB-lpdA) was also studied by adding different concentration of glucose, organic acids (α-ketoglutarate acid, citrate acid and pyruvate acid) in Starkey-S~0 liquid medium. Glucose caused some stimulation on the cell growth of gene engineering strain, butα-ketoglutarate acid and citrate acid showed no effect on the growth of both the wild type and gene engineering strains under the studied concentration level. Pyruvate acid could inhibit the growth of both the wild type and gene engineering strains, but the inhibition could be partially relieved by the gene engineering strains. Although glucose could facilitate the cell growth of A. thiooxidans, but only under the supply of inorganic sulfur, which might indicate the absence of electron transfer system to produce ATP by the metabolism of organic compounds. So, only limited organic compounds could be metabolized by A. thiooxidans.
In conclusion, the genes forα-ketoglutarate dehydrogenase complex were introduced into A. thiooxidans for the first time, and the sucAB and lpdA genes could be expressed in A. thiooxidans under its own promoter. This indicated that the heterotrophic complicated genes for the enzymes (α-ketoglutarate dehydrogenase complex) in the central metabolic pathway could be expressed and assembled in the extremely acidophilic, obligately chemolithoautotrophic A. thiooxidans. Though possessing low enzymatic activities, the constructed gene engineering strains could metabolize glucose more efficient and mitigate the inhibition caused by the metabolic intermediates such as pyruvate acid, and the faster growth strains constructed would be more useful for the application. Our work will not only open a way to investigate the phylogenetic relationship between heterotrophic and autotrophic bateria, but also provide a new insight into how to improve the growth rate and leaching performance of the obligately autotrophic bacteria.
引文
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10.Matin,A.and S.C.Rittenberg,Enzymes of carbohydrate metabolism in Thiobacillus species.J Bacteriol,1971.107(1):p.179-86.
11.Lu,M.C.,A.Matin,and S.C.Rittenberg,Inhibition of growth of obligately chemolithotrophic Thiobacilli by amino acids.Arch Mikrobiol,1971.79(4):p.354-66.
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14.Smith,A.J.,J.London,and R.Y.Stanier,Biochemical basis of obligate autotrophy in blue-green algae and thiobacilli.J Bacteriol,1967.94(4):p.972-83.
15.Butler,R.G.and W.W.Umbreit,Reduced nicotinamide adenine dinucleotide oxidase and alpha-ketoglutaric dehydrogenase activity by Thiobacillus thiooxidans.J Bacteriol,1969.97(2):p.966-7.
16.Hempfling,W.P.and W.Vishniac,Oxidative phosphorylation in extracts of thiobacillus X.Biochem Z,1965.342(3):p.272-87.
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18.Trudinger,P.A.and D.P.Kelly,Reduced nicotinamide adenine dinucleotide oxidation by Thiobacillus neapolitanus and Thiobacillus strain C.J Bacteriol,1968.95(5):p.1962-3.
19.Pan,P.and W.W.Umbreit,Growth of obligate autotrophic bacteria on glucose in a continuous flow-through apparatus.J Bacteriol,1972.109(3):p.1149-55.
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22.Lundgren,D.G.and K.F.Bott,Growth and Sporulation Characteristics of an Organic Sulfur-Requiring Auxotroph of Bacillus Cereus.J Bacteriol,1963.86:p.462-72.
23.Starkey,R.L.,Concerning the Carbon and Nitrogen Nutrition of Thiobacillus Thiooxidans,an Autotrophic Bacterium Oxidizing Sulfur under Acid Conditions.J Bacteriol,1925.10(2):p.165-95.
24.WAKSMAN,S.A.,AND STARKEY,R.L.,On the growth and respiration of autotrophic bacteria.Jour.Gen.Physiol.,1923.5:p.285-310.
25.Borichewski,R.M.and W.W.Umbreit,Growth of Thiobacillus thiooxidans on glucose.Arch Biochem Biophys,1966.116(1):p.97-102.
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28.初立恩,颜望明,王祖农,几种外源有机酸对于氧化硫硫杆菌的生长和氧化硫的影响.东大学学报(自然科学版),198l(4):p.110-117.
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30.Waksman,S.A.and J.S.Joffe,Microorganisms Concerned in the Oxidation of Sulfur in the Soil:Ⅱ.Thiobacillus Thiooxidans,a New Sulfur-oxidizing Organism Isolated from the Soil J Bacteriol,1922.7(2):p.239-56.
31.Rao,G.S.and L.R.Berger,Basis of pyruvate inhibition in Thiobacillus thiooxidans.J Bacteriol,1970.102(2):p.462-6.
32.Tuttle,J.H.and P.R.Dugan,Inhibition of growth,iron,and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.Can J Microbiol,1976.22(5):p.719-30.
33.Tuttle,J.H.,P.R.Dugan,and W.A.Apel,Leakage of cellular material from Thiobacillus ferrooxidans in the presence of organic acids.Appl Environ Microbiol,1977.33(2):p.459-69.
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36.Cobley,J.G.and J.C.Cox,Energy conservation in acidophilic bacteria.Microbiol Rev,1983.47(4):p.579-95.
37.Cox,J.C.e.a.,Transmembrane electrical potential and transmembrane pH gradient in the acidophile Thiobacillus ferrooxidans.Biochemical J.,1979.178:p.195-200.
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53.颜望明;,田.林.刘.刘.张.,大肠杆菌磷酸果糖激酶基因在野生型专性自养嗜酸性氧化硫硫杆菌Tt-Z2中的表达.山东大学学报(理学版),Journal of Shandong University(Natural Science),2004(2).
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2.Starkey,R.L.,Symposium on autotrophy.Ⅰ.Introduction.Bacteriol Rev,1962.26:p.142-4.
3.Starkey,R.L.,Concerning the Physiology of Thiobacillus Thiooxidans,an Autotrophic Bacterium Oxidizing Sulfur under Acid Conditions.J Bacteriol,1925.10(2):p.135-63.
4.Konishi,Y.,S.Asai,and N.Yoshida,Growth Kinetics of Thiobacillus thiooxidans on the Surface of Elemental Sulfur.Appl Environ Microbiol,1995.61(10):p.3617-3622.
5.林建群,彭基斌,颜望明,氧化亚铁硫杆菌基因转移系统研究进展.应用与环境生物学报 Chin J Appl Environ Biol,2001.7(2):p.193-6.
6.Kawaguchi,H.,et al.,Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans.J Biosci Bioeng,2000.90(4):p.459-61.
7.刘缨;,林.颜.林.,硫杆菌的分子遗传学研究.微生物学杂志,JOURNAL OF MICROBIOLOGY,2000.20(1):p.32-34.
8.Butler,R.G.and W.W.Umbreit,Absorption and utilization of organic matter by the strict autotroph,Thiobacillus thiooxidans,with special reference to aspartic acid.J Bacteriol,1966.91(2):p.661-6.
9.Johnson,C.L.and W.Vishniac,Growth Inhibition in Thiobacillus neapolitanus by Histidine,Methionine,Phenylalanine,and Threonine.J Bacteriol,1970.104(3):p.1145-1150.
10.Matin,A.and S.C.Rittenberg,Enzymes of carbohydrate metabolism in Thiobacillus species.J Bacteriol,1971.107(1):p.179-86.
11.Lu,M.C.,A.Matin,and S.C.Rittenberg,Inhibition of growth of obligately chemolithotrophic Thiobacilli by amino acids.Arch Mikrobiol,1971.79(4):p.354-66.
12.Winogradsky,S.,Recherches sur les organismes de la nitrification.Ann.Inst.Pasteur,1890.4:p.760-71.
13.Oginsky,E.L.,and W.W.Umbreit,Introduction to bacterial physiology.2nd ed.Freeman & Co.,1959.
14.Smith,A.J.,J.London,and R.Y.Stanier,Biochemical basis of obligate autotrophy in blue-green algae and thiobacilli.J Bacteriol,1967.94(4):p.972-83.
15.Butler,R.G.and W.W.Umbreit,Reduced nicotinamide adenine dinucleotide oxidase and alpha-ketoglutaric dehydrogenase activity by Thiobacillus thiooxidans.J Bacteriol,1969.97(2):p.966-7.
16.Hempfling,W.P.and W.Vishniac,Oxidative phosphorylation in extracts of thiobacillus X.Biochem Z,1965.342(3):p.272-87.
17.Smith,A.J.and D.S.Hoare,Acetate assimilation by Nitrobacter agilis in relation to its "obligate autotrophy".J Bacteriol,1968.95(3):p.844-55.
18.Trudinger,P.A.and D.P.Kelly,Reduced nicotinamide adenine dinucleotide oxidation by Thiobacillus neapolitanus and Thiobacillus strain C.J Bacteriol,1968.95(5):p.1962-3.
19.Pan,P.and W.W.Umbreit,Growth of obligate autotrophic bacteria on glucose in a continuous flow-through apparatus.J Bacteriol,1972.109(3):p.1149-55.
20.Matin,A.,Organic nutrition of chemolithotrophic bacteria.Annu Rev Microbiol,1978.32:p.433-68.
21.Tabita,R.and D.G.Lundgren,Utilization of glucose and the effect of organic compounds on the chemolithotroph Thiobacillus ferrooxidans.J Bacteriol,1971.108(1):p.328-33.
22.Lundgren,D.G.and K.F.Bott,Growth and Sporulation Characteristics of an Organic Sulfur-Requiring Auxotroph of Bacillus Cereus.J Bacteriol,1963.86:p.462-72.
23.Starkey,R.L.,Concerning the Carbon and Nitrogen Nutrition of Thiobacillus Thiooxidans,an Autotrophic Bacterium Oxidizing Sulfur under Acid Conditions.J Bacteriol,1925.10(2):p.165-95.
24.WAKSMAN,S.A.,AND STARKEY,R.L.,On the growth and respiration of autotrophic bacteria.Jour.Gen.Physiol.,1923.5:p.285-310.
25.Borichewski,R.M.and W.W.Umbreit,Growth of Thiobacillus thiooxidans on glucose.Arch Biochem Biophys,1966.116(1):p.97-102.
26.Pronk,J.T.,et al.,Growth of Thiobacillus ferrooxidans on Formic Acid.Appl Environ Microbiol,1991.57(7):p.2057-2062.
27.Amemiya,K.,Relationship between growth and metabolic activity in the strict chemolithotroph,Thiobacillus thiooxidans.Can J Microbiol,1974.20(12):p.1709-12.
28.初立恩,颜望明,王祖农,几种外源有机酸对于氧化硫硫杆菌的生长和氧化硫的影响.东大学学报(自然科学版),198l(4):p.110-117.
29.Kuenen,J.G.and H.Veldkamp,Effects of organic compounds on growth of chemostat cultures of Thiomicrospira pelophila,Thiobacillus thioparus and Thiobacillus neapolitanus.Arch Mikrobiol,1973.94(2):p.173-90.
30.Waksman,S.A.and J.S.Joffe,Microorganisms Concerned in the Oxidation of Sulfur in the Soil:Ⅱ.Thiobacillus Thiooxidans,a New Sulfur-oxidizing Organism Isolated from the Soil J Bacteriol,1922.7(2):p.239-56.
31.Rao,G.S.and L.R.Berger,Basis of pyruvate inhibition in Thiobacillus thiooxidans.J Bacteriol,1970.102(2):p.462-6.
32.Tuttle,J.H.and P.R.Dugan,Inhibition of growth,iron,and sulfur oxidation in Thiobacillus ferrooxidans by simple organic compounds.Can J Microbiol,1976.22(5):p.719-30.
33.Tuttle,J.H.,P.R.Dugan,and W.A.Apel,Leakage of cellular material from Thiobacillus ferrooxidans in the presence of organic acids.Appl Environ Microbiol,1977.33(2):p.459-69.
34.Vishniac,W.and M.Santer,The thiobacilli.Bacteriol Rev,1957.21(3):p.195-213.
35.Alexander,B.e.a.,The relationship between chemiosmotic parameters and sensitivity to anions and organic acids in the acidophile Thiobacillus ferrooxidans.J.Gen.Microbiol.,1987.133:p.1171-9.
36.Cobley,J.G.and J.C.Cox,Energy conservation in acidophilic bacteria.Microbiol Rev,1983.47(4):p.579-95.
37.Cox,J.C.e.a.,Transmembrane electrical potential and transmembrane pH gradient in the acidophile Thiobacillus ferrooxidans.Biochemical J.,1979.178:p.195-200.
38.Kelly,D.P.,Regulation of chemoautotrophic metabolism.Ⅱ.Competition between amino acids for incorporation into Thiobacillus.Arch Mikrobiol,1969.69(4):p.343-59.
39.Rittenberg,S.C.,The roles of exogenous organic matter in the physiology of chemolithotropic bacteria.Adv.Microbial.Physiol.,1969.3:p.159-96.
40.Clark,C.and E.L.Schmidt,Growth response of Nitrosomonas europaea to amino acids.J Bacteriol,1967.93(4):p.1302-8.
41.Gundersen,K.,Effects of B-vitamins and amino acids on nitrification.Physiol.Plant.,1955.8:p.136-41.
42.Delwiche,C.C.and M.S.Finstein,Carbon and Energy Sources for the Nitrifying Autotroph Nitrobacter.J Bacteriol,1965.90(1):p.102-7.
43.Johnson,E.J.and S.Abraham,Enzymes of intermediary carbohydrate metabolism in the obligate autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus.J Bacteriol,1969.100(2):p.962-8.
44.Williams,P.J.and S.W.Watson,Autotrophy in Nitrosocystis oceanus.J Bacteriol,1968.96(5):p.1640-8.
45.Matin,A.and S.C.Rittenberg,Regulation of glucose metabolism in Thiobacillus intermedius.J Bacteriol,1970.104(1):p.239-46.
46.Inoue,H.,et al.,Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.FEMS Microbiol Lett,2002.214(1):p.127-32.
47.Kelly,D.P.,Influence of amino acids and organic antimetabolites on growth and biosynthesis of the chemoautotroph Thiobacillus neapolitanus strain C.Arch Mikrobiol,1967.56(2):p.91-105.
48.Wakil,S.J.,and D.M.Gibson.,Ⅷ.The participation of protein-bound biotin in the biosynthesis of fatty acid.Biochim.Biophys.Acta,1960.41:p.122-9.
49.Umbreit,W.W.,H.R.Vogel,and K.G.Vogler,The Significance of Fat in Sulfur Oxidation by Thiobacillus Thiooxidans.J Bacteriol,1942.43(2):p.141-8.
50.Levin,R.A.,Fatty acids of Thiobacillus thiooxidans.J Bacteriol,1971.108(3):p.992-5.
51.Adair,F.W.,Membrane-associated sulfur oxidation by the autotroph Thiobacillus thiooxidans.J Bacteriol,1966.92(4):p.899-904.
52.Mahoney,R.P.and M.R.Edwards,Fine Structure of Thiobacillus thiooxidans.J Bacteriol,1966.92(2):p.487-495.
53.颜望明;,田.林.刘.刘.张.,大肠杆菌磷酸果糖激酶基因在野生型专性自养嗜酸性氧化硫硫杆菌Tt-Z2中的表达.山东大学学报(理学版),Journal of Shandong University(Natural Science),2004(2).
54.Martin,P.A.,P.R.Dugan,and O.H.Tuovinen,Plasmid DNA in acidophilic,chemolithotrophic thiobacilli.Can J Microbiol,1981.27(8):p.850-3.
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