Functional Analysis of Tungsten Metabolism in Pyrococcus furiosus

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Title: Functional Analysis of Tungsten Metabolism in Pyrococcus furiosus
Author: Lee, Alice Mei
Advisors: Amy Grunden, Committee Chair
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.
Date: 2010-08-03
Degree: PhD
Discipline: Microbiology

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