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Characterization of the carbon dioxide assimilation pathways in the hyperthermophilic archaeon Pyrobaculum islandicum.

机译：嗜热古细菌Pyrobaculum islandicum中二氧化碳同化途径的表征。

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摘要

Most hyperthermophiles are capable of chemolithoautotrophic metabolism. However, the assimilation of CO2 in hyperthermophiles has only been studied extensively in methanogens and little is known about the physiology, metabolism, and enzymology of this process in other hyperthermophiles. The primary purpose of this study was to characterize CO2 assimilation in Pyrobaculum islandicum, a non-methanogenic, hyperthermophilic archaeon. The results of this study were then tested in other hyperthermophilic, autotrophic Archaea to determine whether the phenomena observed are broadly applied by these organisms.;P. islandicum uses the citric acid cycle in oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow in the cycle is poorly understood. P. islandicum was grown autotrophically, heterotrophically, and mixotrophically with acetate, H2, and low amounts of yeast extract. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum differed significantly under the three different growth conditions suggesting that they are regulated. The results suggest that citrate lyase and AMP-forming acetyl-CoA synthetase, and not ATP citrate lyase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle. Pyruvate synthase activity, which converts acetyl-CoA to pyruvate for biosynthesis, was absent from autotrophically and mixotrophically grown cultures.;The key enzymes of citramalate and 3-hydroxypropionate cycles were measured from P. islandicum cells grown autotrophically and mixotrophically to determine how acetyl-CoA is further assimilated in the absence of pyruvate synthase. The results show that it can use the citramalate cycle for acetate assimilation but not for CO2 assimilation. The pathway for acetyl-CoA assimilation in autotrophically grown P. islandicum remains unknown. Another autotrophic organism investigated was the thermoacidophilic archaeon Acidianus brierleyi. The absence of ATP citrate lyase activity in autotrophically-grown A. brierleyi and the presence of 3-hydroxypropionate cycle enzyme activities in a previous study led to the suggestion that this organism only uses the latter cycle for CO2 assimilation. In this study, all of the 3-hydroxypropionate cycle and reductive citric acid cycle enzyme activities, including ATP citrate lyase activity, were detected but the latter enzyme required covalent modification by an acetylating compound (0.03% acetic anhydrite) for activity. This suggests that A. brierleyi combines the reductive citric acid cycle with the 3-hydroxypropionate cycle for CO2 assimilation. The citric acid cycle enzyme activities were also tested for in the hyperthermophilic, autotrophic archaeon Pyrolobus fumarii whose CO2 assimilation pathway is unknown. Most of the activities were absent; therefore, a pathway other than the reductive citric acid cycle appears to be used for CO2 assimilation in this organism.;In order to better understand the citric acid cycle in Archaea and potentially provide the necessary enzyme for citrate lyase purification and characterization, malate dehydrogenase (MDH), the most common citric acid cycle enzyme found in Archaea, was purified and characterized. Kinetic analysis of recombinant MDH indicates that it is optimized for activity in the reductive direction beyond that found in other characterized archaeal MDHs. There also appears to have been very little lateral gene transfer of MDH in Archaea. The results from these studies provide further insight into primary production by hyperthermophilic Archaea, which could potentially be a vast and important process within subsurface geothermal environments in the absence of sunlight.
机译：大多数嗜热菌能够进行化学自养型代谢。但是，仅在产甲烷菌中对高温嗜热菌中的CO2同化进行了广泛的研究，而在其他高温嗜热菌中，对该过程的生理学，代谢和酶学知之甚​​少。这项研究的主要目的是表征不产甲烷，超嗜热古生菌Pyrobaculum islandicum中的CO2同化。然后，在其他超嗜热，自养古细菌中测试了这项研究的结果，以确定观察到的现象是否被这些生物广泛应用。 islandicum使用柠檬酸循环分别在氧化和还原方向上进行异养和自养生长，但是对循环中碳流量的控制了解甚少。 P. islandicum与乙酸盐，H2和少量酵母提取物自养，异养和杂养生长。在三种不同的生长条件下，参与小岛青霉中心代谢的19种酶中11种酶的活性存在显着差异，表明它们受到调节。结果表明，柠檬酸裂解酶和形成AMP的乙酰辅酶A合成酶，而不是ATP柠檬酸裂解酶，与柠檬酸合成酶相反，可控制柠檬酸循环中碳的流动方向。自养和混养培养物缺乏丙酮酸合酶活性，该酶将乙酰辅酶A转化为丙酮酸以进行生物合成;;从自养和混养的海岛island细胞中检测柠檬酸和3-羟基丙酸循环的关键酶，以确定乙酰-如何在没有丙酮酸合酶的情况下，CoA被进一步吸收。结果表明，它可以利用柠檬酸循环进行乙酸同化，而不能用于CO2同化。自养生长的岛状疟原虫中乙酰辅酶A同化的途径仍然未知。被研究的另一种自养生物是嗜热古生古细菌Acidianus brierleyi。自养生长的A. brierleyi中不存在ATP柠檬酸裂合酶活性，而在先前的研究中存在3-羟基丙酸酯循环酶活性，这提示该生物仅将后一循环用于CO2同化。在这项研究中，检测到了所有3-羟基丙酸循环和柠檬酸还原循环酶的活性，包括ATP柠檬酸裂合酶的活性，但是后一种酶需要通过乙酰化化合物（0.03％的无水醋酸酐）进行共价修饰才能发挥活性。这表明A. brierleyi将还原柠檬酸循环与3-羟基丙酸酯循环结合用于CO2同化。还对高温，自养古细菌Pyrolobus fumarii的柠檬酸循环酶活性进行了测试，其CO2同化途径尚不清楚。大多数活动都不参加；因此，在该生物体中，还原性柠檬酸循环以外的其他途径似乎可用于CO2同化。为了更好地了解古细菌中的柠檬酸循环并潜在地提供柠檬酸裂解酶纯化和表征所需的酶，苹果酸脱氢酶（ MDH）是古细菌中最常见的柠檬酸循环酶，经过纯化和鉴定。重组MDH的动力学分析表明，针对其还原方向的活性进行了优化，超出了其他特征古细菌MDH所发现的范围。在古细菌中，MDH的横向基因转移似乎也很少。这些研究的结果为进一步了解高温嗜热古生菌的初级生产提供了可能，这可能是在没有阳光的情况下在地下地热环境中的一个巨大而重要的过程。

著录项

  • 作者

    Hu, Yajing.;

  • 作者单位

    University of Massachusetts Amherst.;

  • 授予单位 University of Massachusetts Amherst.;
  • 学科 Biology Microbiology.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2008
  • 页码 117 p.
  • 总页数 117
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 微生物学;生物化学;
  • 关键词

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