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

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.