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Project Progress

This project report reports on the accomplishments of the project “The evolution and diversity of ancient CO₂ -fixation pathways in anaerobic and extremophilic microorganisms: Clues to the early evolution of life on Earth” as part of NNA04CC04A “From Early Biospheric Metabolisms to the Evolution of Complex Systems” (Stefan Sievert — Co-PI, Woods Hole Oceanographic Institution ( WHOI)).

We have selected a total of 13 bacteria or archaea on the basis of having, or being suspected of having, enzymes of the reductive tricarboxylic acid cycle (RTCA). Most of the microorganisms were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) as active or freeze-dried cultures. Except for two organisms, all are thermophiles that have optimum growth temperatures from 60—100 °C. Incubations at 8 different temperatures within this range were required. Ten different growth media were prepared in serum bottles to re-establish growing cultures from the purchased ones. Since enzymes involved in autotrophic CO₂ fixation can be oxygen sensitive, a procedure to harvest and store cells under anoxic conditions was developed and employed. Cells for nucleic acid extraction were also concentrated and stored by the same method. So far, nucleic acids have been extracted from eight cultures. We have also received deoxyribonucleic acid (DNA) from Constantino Vetriani (Rutgers University) and Ken Takai (JAMSTEC, Japan), with whom we are collaborating on these questions. All these organisms either belong to the ε-proteobacteria or the Aquificales . Using a PCR protocol established in our laboratory we have so far successfully amplified ACL genes from eight cultures. In addition, we are also in the process of obtaining ACL sequences from deep-branching eukaryotes to reconstruct the evolutionary history of this enzyme. So far we have amplified ACL from representatives from seven groups.

We now have biochemical and genetic evidence for the operation of the RTCA in two members of the ε-proteobacteria , and in Desulfurobacterium autotrophicum ( Aquificales ). Bacteria of the ε-proteobacteria have been identified as very important members of the microbial community at deep-sea hydrothermal vents. However, no information exists as to what CO₂ -fixation pathway might be used by ε-proteobacteria or Desulfurobacterium . This work is presently being prepared for submission to the Journal of Bacteriology .