Regulation of nitrogen fixation in Rhodospirillum rubrum : Through proteomics and beyond

Detta är en avhandling från Stockholm : Department of Biochemistry and Biophysics, Stockholm University

Sammanfattning: Adaptability is one of the reasons for the success of bacteria, allowing them to survive in conditions where no other organisms would be able to thrive. Nitrogen deficiency, for example, can be a limiting factor for the growth of micro-organisms, as this element is an essential part of almost all types of biomolecules. As such, some bacteria have evolved specific mechanisms to overcome nitrogen limitation. Nitrogen fixing bacteria, or diazotrophs, use a specific enzyme complex, nitrogenase, in order to harness this element from the enormous reservoir that is the Earth’s atmosphere. However, nitrogen fixation is a demanding process for the cells, requiring vast amounts of energy and tight regulation.In this thesis we explore the mechanisms regulating nitrogen fixation in Rhodospirillum rubrum, a purple non-sulphur photosynthetic bacterium. Using proteomics tools, we show how the regulation of both the nitrogen and carbon fixation processes is interconnected, possibly in order to maintain the intracellular redox balance. Using a new detergent molecule, we also demonstrate how nitrogen availability affects the chromatophore membrane proteome.Our studies have revealed the crucial role of the cellular pool of 2-oxoglutarate (2OG) for adequate signaling through the PII proteins and the effects resulting from artificially manipulating this metabolite’s concentration. In R. rubrum nitrogenase is also subjected to post-translational control (the “switch-off” effect) and this work shows for the first time that the enzyme modifying nitrogenase (Dinitrogenase Reductase ADP-ribsosyl Transferase or DRAT) forms a complex with the PII protein GlnB. This complex allows DRAT activation and its formation – and, therefore, DRAT activity – is regulated by binding of ADP:ATP and 2OG to GlnB.Upon light withdrawal, nitrogenase activity anaerobically in the dark is also here demonstrated to be dependent on the activity of the pathway starting in pyruvate formate-lyase and we show how different nitrogen sources influence the switch-off response. This response can, in some conditions, be modified by addition of pyruvate and we have studied how this metabolite influences nitrogenase activity and switch-off regulation.This study allows a better understanding of the underlying processes controlling the metabolic routes in R. rubrum and also provides new insights into regulation of enzyme activity, paving the road for the complete establishment of the mechanisms regulating nitrogenase switch-off.

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