Date of Completion

5-8-2015

Embargo Period

11-4-2015

Keywords

Symbiosis, Central Metabolism, PTS, Nitrogen Regulation

Major Advisor

Daniel Gage

Associate Advisor

Victoria Robinson

Associate Advisor

David Benson

Associate Advisor

Carolyn Teschke

Associate Advisor

Joerg Graf

Field of Study

Microbiology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Sinorhizobium meliloti is a soil bacterium capable of forming an intracellular symbiosis with temperate legumes. During symbiosis S. meliloti will fix atmospheric nitrogen, which it provides to its host plant. In return for fixed nitrogen, the plant provides its microsymbiont with carbon in the form of C4-dicarboxylates. Unlike most model organisms, S. meliloti prioritizes catabolism of succinate, a phenomenon referred to as succinate-mediated catabolite repression (SMCR). SMCR is controlled by an incomplete phosphotransferase system (PTS), which, unlike carbohydrate-type PTS, is a regulatory system that is not involved in sugar transport. This work uses a biochemical approach to elucidate the signals involved in regulating PTS activity.

Biochemical characterization of S. meliloti EINtr revealed that the enzyme is inhibited by glutamine, a major signal of nitrogen availability in proteobacteria. EINtr detects glutamine through its N-terminal GAF domain, a ubiquitous small molecule binding domain. In contrast to E. coli EINtr, the S. meliloti enzyme is not activated by a-ketoglutarate. The differences in EINtrregulation likely reflect the preferred carbon source of each organism, with glucose entering central metabolism through glycolysis and succinate entering through the TCA cycle, sharing a metabolic pathway with a-ketoglutarate. Phosphorylated HPr-His in S. meliloti is much less stable than E. coli P~His-HPr, and this instability is due to an arginine residue that is conserved within a-proteobacteria that only contain an incomplete PTS. Rapid phosphohydrolysis of P~His-HPr in the a-proteobacteria may be act to remove phosphate from the system to avoid oversaturation, a problem that is not faced by sugar-phosphorylating PTS. The work presented here sheds light on how the PTS of S. meliloti integrates carbon, nitrogen, and energy levels within the cell in order to regulate SMCR.

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