Date on Master's Thesis/Doctoral Dissertation

12-2015

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Perlin, Michael

Committee Co-Chair (if applicable)

Graham, James

Committee Member

Graham, James

Committee Member

Schultz, David

Committee Member

Steffen, Joseph

Committee Member

Worley, Micah

Author's Keywords

nitrogen cycle; corn smut; nitrogen fixation; stable isotope; SIRMS

Abstract

Nitrogen is an essential nutrient for all living creatures. Ammonium is one of the most efficiently used and thus preferred, sources of nitrogen. As with other dimorphic fungi, yeast-like cells of Ustilago maydis, a fungal pathogen of maize, switches to filamentous growth when starved for nitrogen/ammonium. U. maydis carries two genes, ump1 and ump2, encoding ammonium transporters that facilitate both uptake of ammonium and the filamentous response to its absence. While no obvious phenotype is observed when ump1 is deleted, cells without ump2 are unable to filament in response to low ammonium, although they can still grow. Surprisingly, ump1ump2 double mutants can also grow on low ammonium. More amazing still, both wild-type and mutant cells continue to grow, even after strenuous efforts were made to remove all nitrogen sources from their growth media. To further investigate these unusual observations, we examined the growth character of cells in various low and no-ammonium conditions with variable glucose concentrations, examined isotopic enrichment employing 15N2 gas as a tracer and D-[U-13C]Glucose, conducted PCR screenings and evaluated the possibility of an endosymbiont. The Dump1Dump2 mutant appeared to produce longer cells than the wild-type and achieved higher titers under 50 mM glucose with no ammonium. That mutant also incorporated more 15N than the wild-type in liquid culture under low and no-ammonium conditions. Cells passed through serial treatments of high levels of antibacterial compounds persisted in growth and 15N accumulation. PCR results indicated there was neither prokaryotic 16S rDNA nor the gene for dinitrogenase reductase, typical of prokaryotic diazotrophs. Overall our studies indicate the novel discovery of an unknown nitrogen fixation system in U. maydis. While the molecular mechanism remains unresolved a metabolic capacity to convert dinitrogen into nitrogen that is bioavailable natively in a eukaryotic system holds the potential to change our understanding of the biogeochemical cycling of nitrogen. Moreover, these findings also present a potential way to reduce the anthropogenic contribution of organic nitrogen that is a large contributor to the accretion of eutrophication, “dead zones”, in our coastal waters and large lake system.

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