Browsing by Author "Kalis, Katie"
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Item Molecular manipulations of Cba. tepidum to investigate sulfur oxidation and energy regulation(University of Delaware, 2019) Kalis, KatieChlorobaculum tepidum is a model organism for anaerobic phototrophic sulfur oxidation and is unusual amongst other microbes that interact with insoluble minerals in that it can both produce and subsequently consume extracellular S(0); however, the enzymes that catalyze S(0) production and consumption are thought to reside in the cytoplasm, periplasm, or membranes of the cell. The mechanism and proteins involved in the formation and transport of biogenic S(0) across the outer membrane during production and consumption are unknown. Differential expression of Cba. tepidum genes during S(0) production and consumption relative to growth on thiosulfate revealed membrane protein candidates that may play a role in biogenic S(0) metabolism. Mutants lacking candidate genes were tested for defects during growth on sulfide, thiosulfate, and sulfur. To date, these mutant strains have displayed no significant growth defects. These results indicate that there may be downstream regulation of expressed genes involved in S(0) metabolism or multiple pathways for the formation and transport of S(0). In addition, the localization of proteins involved in S(0) metabolism is unknown. Interestingly, shotgun proteomics of S(0) globules revealed a subset of proteins expressed during S(0) production. Yellow Fluorescence-Activating and Absorption Shifting Tag (Y-FAST) is a fluorescent label used to visualize the subcellular or extracellular location of proteins fused to Y-FAST. An attempt to develop this technique in Cba. tepidum is presented here. Further development may lead to the successful localization of recombinant proteins from the S(0) globule protein subset and serve as a useful tool to localize other recombinant proteins in Cba. tepidum in future research. Additionally, the work presented here aided in the successful development Y-FAST in a different anaerobe, Clostridium acetobutylicum. Biogenic S(0) metabolism involves the processing and export of S(0) and proteins, which is energetically costly. Therefore making and consuming S(0) likely causes an energy stress. As part of a larger project on energy stress in prokaryotes, global responses to energy metabolism were examined to determine if DNA modification might play a role in energy stress responses.Item Nitrate reduction to ammonium in Nautilia profundicola via a proposed reverse HURM pathway(University of Delaware, 2013) Kalis, KatieNautilia profundicola is an epsilonproteobacterium that inhabits hydrothermal vents on the dorsal epithelium of Alvinella pompejana or as a free-living bacterium. N. profundicola was isolated with polysulfide as the electron acceptor for respiratory growth, but is also able to utilize nitrate as an electron acceptor for growth. Interestingly, the N. profundicola genome lacks homologs of known enzymes used in nitrite reduction. Therefore, the pathway of nitrate reduction is likely to be novel. Genome annotation suggested that the reverse hydroxylamine ubiquinone redox module (rHURM) may be used for anaerobic cellular respiration in N. profundicola when nitrate is the terminal electron acceptor. The proposed rHURM pathway includes rHaoA and hcp/har which catalyze the reduction of nitrite to hydroxylamine, as an intermediate, and hydroxylamine to ammonium, respectively. In this study, cell growth was monitored in batch cultures under varied concentrations of sulfide, nitrate, formate, and H2. Optimal concentrations were used for culturing cells in continuous culture and in batch culture for substrate addition assays. The four nitrogen compounds in the proposed pathway were measured in the substrate addition assays—nitrate, nitrite, hydroxylamine, and ammonium. Hydroxylamine was present in small concentrations during nitrate reduction to ammonium. When given as a substrate, hydroxylamine was directly and entirely reduced to ammonium (up to 5 mM). An anaerobic continuous culture apparatus for N. profundicola was developed to grow cells in steady state with different electron acceptors to eliminate growth rate variations that have affected prior gene expression measurements. The results presented here are consistent with hydroxylamine as a free intermediate in nitrate reduction to ammonium in N. profundicola. Additional experiments including in vitro assays of enzymatic activity and gene disruption will be required to fully address the role of the proposed rHURM pathway in nitrate reduction.