Date of Award

8-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Committee Member

Barbara Campbell, Committee Chair

Committee Member

Mike Henson

Committee Member

Harry D Kurtz

Abstract

Recent genomic studies within the Epsilonproteobacteria have uncovered a potentially novel mechanism for chemotrophic respiration: the reverse Hydroxylamine Ubiquinone Redox Module (rHURM), a dissimilatory nitrate reduction pathway utilizing a hydroxylamine intermediate. Originally discovered in the chemoautolithotroph Nautilia profundicola, genes indicative of the rHURM pathway have been identified in several species of Campylobacter, including C. curvus. In the absence of classic nitrite reductase genes, a hydroxylamine oxidoreductase (hao) homolog encodes a periplasmic octoheme potentially capable of reducing nitrite, a product of periplasmic nitrate reductase (NapA), to hydroxylamine, which is then converted to ammonium by a putative hydroxylamine reductase hybrid cluster protein (Hcp). This research assesses the expression of these genes in nitrate amended cultures compared to fumarate amended cultures. Our results suggest that all three core nitrogen metabolic genes were up-regulated in cells grown in the presence of nitrate as opposed to the fumarate, during mid log phase growth. The hao gene exhibited the highest degree of expression of all three genes, 15 fold greater than hcp. However, no significant difference in hao expression was detected at early log phase growth between nitrate and fumarate respiring cells. By performing growth curves with induced reactive nitrogen species (RNS) stress, we observed that nitrate respiring cells were resistant to the toxic effects of exogenous hydroxylamine compared to fumarate respiring cells, yet both cultures exhibited similar resistance to sudden nitrite additions. This evidence suggests that the core genes involved in the putative rHURM pathway are actively expressed in the presence of nitrate, however Hao may have an alternative function or a unique method of regulation compared to nitrate and hydroxylamine reductase. Future research may include analyzing transcriptomes to better characterize expression of associated genes and regulatory pathways involved in nitrogen metabolism.

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