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Title: The enigmatic Calvin cycle in chemoautotrophic symbionts
Abstract: Symbiosis is a major driving force of biological diversity. Organism within mutualistic symbiotic associations are capable of occupying new ecological niches which would have been otherwise inaccessible to their individual partners. Symbioses between chemoautotrophic bacteria and marine invertebrates are an example of such partnerships in which hosts benefit from organic carbon supplied by its symbiotic bacteria, while the symbionts profit from a steady supply of reduced inorganic compounds and electron acceptors sequestered and delivered to them by their hosts. These symbioses are able to, for instance, create lush oases of life surrounding hydrothermal vents in the deep sea amid the otherwise barren seabed. Perhaps the most enigmatic feature shared by all chemoautotrophic symbionts within the class of gammaproteobacteria in the lack for a gene encoding fructose 1,6-bisphosphatase (FBPase), an enzyme which in bacteria catalyzes two essential reactions in the Calvin-Benson-Bassham (Calvin) carbon fixation cycle. Yet chemoautotrophic bacterial symbionts are not only able to fix CO2 using the Calvin cycle, but are among some of the most prolific primary producers in the ocean. It has been hypothesized that a glycolytic pyrophosphate-dependent phosphofructokinase (PPi-PFK), acting in reverse, can perform the function of the missing FBPase in these bacteria. To test this hypothesis in my thesis I investigated the ability of PPi- PFK from the symbionts of Solemya velum coastal protobranch bivalve to perform the biochemical function of the missing FBPase. I detected high gene expression of the symbiont PPi-PFK-encoding gene and high reverse PPi-PFK activity in the symbiont-containing tissue of the host. The recombinant enzyme for the S. velum symbiont had the highest specificity for the reverse reaction compared to other bacterial PPi-PFKs and higher catalytic efficiency than many bacterial FBPases. By recreating the symbiont-like Calvin cycle in a free-living closely-related purple sulfur gammaproteobacterium, Allochromatium vinosum, I demonstrated that in the absence of FBPase its function in the cycle can be performed by PPi-PFK. The shift from FBPase to PPi-PFK in A. vinosum came at the cost of reduced growth and decreased adaptability but offered an improvement in thermodynamic efficiency potentially due to metabolism of the high energy pyrophosphate generated by PPi-PFK in the Calvin cycle. Data presented in my thesis show that the selection of PPi-PFK over FBPase took place in all lineages of gammaproteobacterial chemoautotrophic symbionts. My results also demonstrate that PPi-PFK can perform the biochemical function of FBPase and may have become specifically adapted to this function in the symbionts. The feasibility of the Calvin cycle which uses PPi-PFK instead of FBPase was demonstrated in A. vinosum. The observed physiological changes accompanying the shift from FBPase to PPi-PFK in this bacterium suggest that such a transition could be advantageous to the symbionts. Living in a relative constant and isolated host environment, these specialist bacteria may be selected for the thermodynamic efficiency which accompanies PPi-PFK use. Free-living bacterial generalists, on the other hand, would be severely disadvantaged by the associated decline in growth rate and adaptability, as they would become less fit to outgrow their competition and slower at adapting to fluctuation in environmental conditions. A proposed link between PPi-PFK reverse activity in the Calvin cycle and a sulfur oxidation pathway in chemoautotrophic symbionts may explain why a shift to PPi- PFK has not occurred in photoautotrophic symbionts and plasmids, which obtain their energy from light instead of sulfide. These results advance our understanding of the key metabolic processes and evolutionary forces responsible for the origin and maintenance of chemoautotrophic symbioses.
Committee: Colleen Cavanaugh (Advisor), Peter Girguis, Hopi Hoekstra