MIP: Denitrification: Not Just for Prokaryotes Anymore?

Woods Hole Oceanographic Institution, Woods Hole MA

Investigators

Abstract

The process of denitrification, which occurs in anoxic (i.e., lacking oxygen) or micro-oxic environments, is defined as the conversion of nitrate or nitrite to gaseous products such as nitrogen and/or nitrous oxide. This process is believed to be the primary mechanism for removing bioavailable nitrogen from the sea and thus plays an important role in the "fertility" of the ocean on long time scales. The genetics and biochemistry of denitrification has been studied in Bacteria and Archaea, as well as two species of Eukaryotic fungi. Now, according to a recent study, certain benthic foraminiferan protists are capable of complete denitrification. This is an important finding, which suggests an expanded capability for denitrification in the Eukaryotic domain, although the mechanisms of foraminiferal denitrification have not yet been identified. The broad objective of this project is to examine the metabolic capabilities and microbial associations in certain foraminifera that allow these foraminiferal species to denitrify. Three foraminiferal species will be analyzed: one that is known to sequester nitrate-reducing chloroplasts; one that is novel (i.e., undescribed) but is known to have bacterial endsymbionts associated with its cytoplasmic vacuoles; one that does not possess any endosymbionts. This project will test the following hypotheses: 1) vacuoles in the foraminiferal cytoplasm store nitrate, which can be respired by the endobionts or the foraminiferal host, and (2) denitrification is aided by endosymbionts within the novel foraminifer. The following specific aims will be addressed: (1) identification of the microbial endosymbionts associated with the novel foraminifer using sequence analyses and fluorescent in situ hybridization (FISH); (2) identification and localization of gene products associated with denitrification in foraminifera using mRNA-FISH; (3) survey for intracellular nitrate accumulation using hot vanadium chloride assays; (4) determination of the elemental composition (C, N, S) of foraminiferal vacuoles using High Pressure Freezing, Freeze Substitution, and correlative High Voltage Electron Microscopy (HVEM) and Secondary Ion Mass Spectrometry (SIMS); (5) measurement of rates of foraminiferal denitrification. Results will enable better understanding of microbial affects on benthic anoxic and micro-oxic oceanic ecosystems, as well as global nutrient cycling. The project will also increase understanding of the functioning of protist-prokaryote symbioses and may help to assess their potential impact on eukaryotic ecology and evolution. This project has varied educational components, ranging from middle school to undergraduate studies, and outreach opportunities for secondary school teachers and the lay-public.