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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”. (Dr. Stefan Sievert — Co-PI, WHOI)

We have amplified and sequenced the ATP citrate lyase gene, a key enzyme of the reductive TCA cycle (rTCA) used by certain chemolithoautotrophic microorganisms for autotrophic carbon fixation, from a variety of epsilon-proteobacteria (seven in total) as well as members of the Aquificales (six in total). By using additional, newly designed primers we have in many cases sequenced a gene fragment that includes the gene encoding for the small subunit and the large subunit of ATP citrate lyase as well as the region between the genes. This longer sequence will significantly improve our phylogenetic analyses aimed at reconstructing the evolutionary history of this gene and potentially the whole pathway. The data obtained so far support the view of the rTCA as an ancient metabolic pathway from which other pathways could have evolved and the usefulness of the ATP citrate lyses gene as a functional gene marker. Additionally, we have tested for the activities of key enzymes of the different carbon fixation pathways in seven chemolithoautotrophic organisms, and could further confirm that epsilon-proteobacteria and members of the Aquificales generally use the reductive TCA cycle for autotrophic carbon fixation. This has important ecological implications as members of these groups are important components of microbial communities in hydrothermal systems (both terrestrial and marine). We have also started analyzing environmental samples obtained from deep-sea hydrothermal vents and the Rio Tinto for the presence and diversity of organisms utilizing the rTCA using the ATP citrate lyase gene as a functional gene marker. In addition, the Co-PI successfully nominated Thiomicrospira denitrificans, a chemolithoautotrophic sulfur-oxidizing epsilon-proeteobacterium that uses the reductive TCA cycle for carbon fixation, for whole genome sequencing at JGI through DOE. In the meantime, the genome is finished and we are currently involved in annotation. Having the genome available will prove invaluable for further evolutionary and physiological studies, ranging from comparative genomics to gene expression studies.

Meanwhile, our work demonstrating the operation of the reductive TCA cycle for autotrophic carbon fixation for the first time in epsilon proteobacteria has now been published in the Journal of Bacteriology (Vol. 187: 3020-3027). We are currently in the process of preparing two manuscripts for submission based on the work performed over the last year.