Functional Analysis of Tungsten Metabolism in Pyrococcus furiosus

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dc.contributor.advisor Amy Grunden, Committee Chair en_US Lee, Alice Mei en_US 2010-08-19T18:14:08Z 2010-08-19T18:14:08Z 2010-08-03 en_US
dc.identifier.other etd-07082009-142918 en_US
dc.description.abstract The anaerobic hyperthermophilic archaeon Pyrococcus furiosus is reported to require the metal tungsten for growth. The metal is bound to a unique tricyclic pterin moiety to form the tungstopterin, part of the active center in tungstoenzymes from this organism. The study of tungsten metabolism in P. furiosus has mostly been confined to cultivation studies and the purification and characterization of tungstoenzymes. Given the importance of this element in some biological systems, such as in the model hyperthermophile organism P. furiosus, a functional genomics approach to investigate tungsten metabolism in P. furiosus was undertaken. The use of technologies such as whole genome microarray analysis and the development of proteome profiling techniques enable a systems level understanding of tungsten homeostasis in this important extremophile. We investigated the role of several putative tungsten cofactor biosynthesis genes by using in vivo and in vitro complementation studies in Escherichia coli, a genetically tractable bacterium that has a homologous molybdenum cofactor synthesis system. The results of this study indicate that there is conservation between the tungsten and molybdenum cofactor biosynthetic pathways, as evidenced by the partial in vivo complementation of one of the moeA homologs in P. furiosus. Next, metal homeostasis was explored using whole genome microarray analysis of chemostat cultures of P. furiosus in response to limiting, standard and high concentrations of tungsten. The data analysis indicated that the transcriptomic response in P. furiosus cultured with standard (10 μM) compared to no added (≤ 30 nM) tungsten are nearly identical. However, the transcriptome under high (0.5 mM) when compared to standard and no added tungsten indicated that transcriptional adjustments occurred, primarily in genes related to amino acid metabolism and the transport of inorganic ions. Surprisingly, genes important in tungsten metabolism were minimally impacted by varying concentrations of tungsten. Lastly, we have developed a simple shotgun proteomics approach for the global identification of the P. furiosus proteome. This methodology enabled the identification of over 900 proteins, representing ~44% of the proteome. This is the first high throughput method developed for handling of hyperthermophilic proteomes and will enable extensive proteomics data to be obtained for this organism. en_US
dc.rights I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dis sertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. en_US
dc.subject archaea en_US
dc.subject shotgun proteomics en_US
dc.subject tungsten en_US
dc.subject hyperthermophiles en_US
dc.subject pyrococcus furiosus en_US
dc.subject microarray en_US
dc.title Functional Analysis of Tungsten Metabolism in Pyrococcus furiosus en_US PhD en_US dissertation en_US Microbiology en_US

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