Abstract:

Shewanella oneidensis MR-1 is an attractive candidate for bioremediation because of its metabolic versatility. It has the capacity of utilizing a wide range of terminal electron acceptors in respiration, including toxic metals, such as uranium and chromium. Based on previous biochemical studies and recent complete genome sequence, several unusual features of S. oneidensis metabolism have been investigated but not rigorously verified by 13 C tracer experiments. In order to engineer or modify the behavior of S. oneidensis MR-1 to make it effectively in bioremediation strategies, it is important to gain a complete picture of intracellular metabolic pathways.

In the first part of this dissertation, the distributions of intracellular metabolic fluxes in S. oneidensis MR-1 under aerobic conditions is presented. 13 C-labeled lactate was fed to MR-1, and nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GC-MS) are coupled to analyze isotopomers of proteinogenic amino acids. The intracellular metabolic flux distributions under both aerobic and microaerobic conditions were determined and several active pathways were identified. The findings provide us the first insights into the general metabolism in MR-1 under aerobic conditions.

Ability to use free amino acids as carbon sources is also widespread in MR-1. Therefore, it will be interesting to probe specific metabolic pathways in MR-1 by using 13 C-labeled amino acids. It is relevant to understand the amino acid transport mechanisms before targeting the specific metabolic pathways. In the second part of this dissertation, investigation of amino acid transports in S. oneidensis MR-1 is presented. One- and two-dimensional NMR spectroscopy is used to analyze transport behavior of amino acids. Four different utilization categories with respect to lactate usage were found when supplementing with a single amino acid. When a mixture of sixteen amino acids was fed to the cells, several amino acids shared common transport mechanisms while others used specific transport mechanisms. In the last part of this dissertation, the secretion of alanine by MR-1 and its relationship to glycine and pyruvate is described. The knowledge gained from the studies described in this dissertation will contribute to the overall understanding of the metabolic network of Shewanella oneidensis MR-1.