Degree Type

Dissertation

Date of Award

2014

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Basil J. Nikolau

Abstract

In this dissertation three studies are presented, all of which incorporated metabolomics as part of their experimental methods. The first study is a loss of function study focused on examining E. coli ΔfabH and ΔfabF single and double mutants. Metabolomics studies of ΔfabH β-Ketoacyl-acyl carrier protein synthases (KAS) III (KASIII) assisted in the development of the hypothesis that fabF (KASII) is involved in the survival of ΔfabH E. coli cells. This hypothesis was tested by generating and evaluating ΔfabF/ ΔfabH double mutants. Characterization of the ΔfabF/ ΔfabH double mutant revealed that the fabB gene (KASI) is able to support E. coli cell growth as the sole KAS enzyme present in this strain. Additionally it was determined that fabF is required for the hyper-accumulation of cis-vaccenic acid observed in ΔfabH mutants.

The second study focused on a functional genomics effort to develop a gene-function annotation method based on metabolomics and NMR ligand binding assays. This study had mixed results. The metabolomics effort in this study was met with great technical hurdles. Only in a single case (MA3250) was metabolomics useful in annotating a gene's functions. While the NMR substrate binding assays were able to provide useful data for more accurate gene functional annotations in more than 10% of the genes examined.

The third study presented was an in depth study of the biotin network of Methanosarcina acetivorans, where metabolic changes associated with the presence and absence of biotin in Methanosarcina acetivorans cultures was investigated. In this study metabolomics studies were able to detect metabolic changes that were associated with the presents or absence of biotin. Additionally it was revealed that M. acetivorans does not require biotin or any detectable biotinylated protein to survive and maintain growth.

Copyright Owner

Lucas John Showman

Language

en

File Format

application/pdf

File Size

165 pages

Included in

Biochemistry Commons

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