Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Kathleen Scott, Ph.D.

Committee Member

Valerie Harwood, Ph.D.

Committee Member

Mya Breitbart, Ph.D.

Committee Member

Stephen Deban, Ph.D.

Keywords

autotrophy, carbon fixation, chemotrophy, Hydrothermal vents, Riftia, symbiosis

Abstract

The siboglinid tubeworm Riftia pachyptila is a dominant member of the deep-sea megafauna where seawater and hydrothermal vent (HTV) effluent interface and mix. It is one of the fastest growing invertebrates on land or in the sea. It does not have a digestive tract (e.g. mouth, gut, or anus), and is completely dependent on its sulfur-oxidizing endosymbiont, the Gammaproteobacterium “Candidatus Endoriftia persephone” for its nutritional requirements. This association was the first and is the most well studied among chemolithoautotrophic symbioses. “Ca. E. persephone” is a chemolithoautotrophic bacterium that oxidizes sulfide as an electron donor for energy, reduces oxygen as a terminal electron acceptor, and uses dissolved inorganic carbon for biosynthesis. The symbionts are intracellular, and inhabit the host’s trophosome organ. The symbionts are supplied with sulfide, oxygen, and dissolved inorganic carbon via host blood, and the products of carbon fixation by “Ca. E. persephone” are translocated to the host for growth.

The hydrothermal vent environment is chemically heterogeneous over varying time scales. Substrates required for this symbiotic association are sometimes unavailable from minutes to hours to days at a time. Yet, the giant tubeworm maintains a growth rate of 1.5 meters in less than 2 years. The research presented here investigated adaptations that “Ca. E. persephone” might have to flourish under conditions of habitat heterogeneity. These potential adaptations had been suggested by -omics studies, and here I used biochemical and physiological measurements to investigate them. Three hypotheses were investigated: that the less energetically expensive reductive citric acid cycle (rCAC) is more active when sulfide is less available in the environment, that hydrogen can be used as an electron donor when sulfide concentrations are low, and that the symbiont can supplement oxygen as a terminal acceptor with nitrate.

The first hypothesis that I tested was based on the presence of genes encoding two autotrophic carbon fixing pathways: the Calvin-Benson-Bassham cycle (CBB) and the reductive citric acid cycle (rCAC). I hypothesized that the rCAC would be preferentially activated under low-sulfide conditions, as its energetic requirements are lower, while the CBB would be preferentially activated under high-sulfide conditions. Using enzyme assays that are diagnostic of these pathways, no difference in overall carbon fixation rates, in the activity of either RuBisCO activities or ATP citrate lyase activities was detected from worms incubated in high sulfide versus low sulfide conditions. Electron donor availability does not appear to influence the differential expression of either carbon fixation pathway.

Hydrogenase genes have been detected in the “Ca. E. persephone” genome, and the role of H2 as a major electron donor for the symbiont has been proposed based on the presence of these genes, but not biochemically demonstrated. Based on these findings, I hypothesized that H2 could be used as an electron donor for energy by the symbiont. Using measurements of the effect of H2 on carbon fixation rates and hydrogenase assays by trophosome homogenate, I investigated the role of hydrogen as a major electron donor for this symbiosis. Carbon fixation by trophosome homogenates was not stimulated in the presence of hydrogen and hydrogenase activity was not detectable in trophosome samples. Based on these results, hydrogen does not appear to be a major electron donor for this symbiosis.

Nitrate has been proposed as an alternative electron acceptor for the symbiont when oxygen concentrations are low, and as a source of nitrogen for the host. There is evidence that “Ca. E. persephone” can reduce nitrate to nitrite via nitrate reductase based on incubation experiments, and genomic, transcriptomic, and proteomic data. I hypothesized that “Candidatus Endoriftia persephone” can utilize nitrate to supplement oxygen as a terminal electron acceptor. I measured the effect of nitrate on the rate of carbon fixation in trophosome homogenate and we measured the activity of nitrate reductase in the symbiont. I observed that nitrate stimulates carbon fixation in the trophosome. Nitrate reductase activity was detected in homogenized trophosome samples, but not in R. pachyptila vestimentum, which does not contain symbionts. Clearly, nitrate plays a role in symbiont metabolism, but its contribution to either respiration or biosynthesis remains unresolved.

The results of the experiments reported here suggest that symbiont carbon fixation pathways are not differentially active, but constitutively active in low and high sulfide environments, that H2 is not an electron donor, but that detected hydrogenases could instead regulate intracellular redox in the symbiont, and that nitrate reductase catalyzes the bacterially mediated reduction of nitrate to nitrite. Our experiments failed to support assertions made by -omics studies and underscore the importance of physiological experiments to support hypotheses suggested by -omics data. While –omics studies detect the presence of genes, measure transcript quantities, or demonstrate the presence of a protein, functional capabilities in an organism must be confirmed via biochemical and physiological measurements.

Download

Share

COinS