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Project Progress

We have hypothesized that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in the extreme environments of hydrothermal vents. Autotrophic carbon fixation was characterized in representative members of three lineages of the bacterial phylum Aquificae. Aquificae plays an important role in biogeochemical processes in a variety of high temperature habitats. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO₂ fixation. This is the first time that strains of the Hydrogenothermaceae and 'Desulfurobacteriaceae' have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and 'Desulfurobacteriaceae' use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Thus, these findings substantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is a significant pathway hydrothermal habitats.

We have further analyzed samples from deep-sea vents for the presence and diversity of organisms utilizing the rTCA cycle. We are specifically interested in microorganisms colonizing newly exposed surfaces. A major objective of this research is to understand biological/geochemical interactions during initial colonization of basalt at deep-sea hydrothermal vents through time-series studies that combine molecular genetic characterization of colonists and in situ measurements of fluid chemistry. Our hypothesis is that autotrophic microbes will be the first colonizers, followed by increasing numbers of heterotrophic microorganisms over time. We could detect a high diversity of ACL-genes on basalt panels set out for colonization, suggesting that autotrophic organisms utilizing the rTCA cycle are likely to be early colonizers.

We have annotated the genome of Thiomicrospira denitrificans, which represents the first chemolithoautotrophic bacterium belonging to the ε-proteobacteria that has been sequenced. Its genome provides a reference point for similar organisms that have been identified as important members in many different environments characterized by redox interfaces. Submission of a manuscript is expected within the next three months.

We contributed critical data showing that the symbionts of the deep-sea hydrothermal vent tubeworm Riftia pachyptila, which have been considered a prime example for carbon fixation via the Calvin cycle, also utilize the rTCA cycle. The possible occurrence of two carbon fixation pathways in one organism is unprecedented, finally providing an explanation for the longstanding dilemma that the stable carbon isotopic composition of Riftia is substantially heavier than would be expected by the use of the Calvin cycle alone.