UBC Theses and Dissertations
Isolation and regulation of expression of the Rhodobacter capsulatus suca gene encoding the E1o component of the α-ketoglutarate dehydrogenase enzyme complex Dastoor, Farahad
The citric acid cycle (CAC) is an extremely wide-spread metabolic pathway involved in energy production and intermediary metabolism. One of the key enzymes of the CAC is the α-ketoglutarate dehydrogenase (KGD) enzyme complex. In facultative bacteria, activity of the KGD enzyme complex appears to vary in keeping with the primary function of the CAC, in response to growth conditions (energy production or biosynthesis). Therefore, under anaerobic conditions synthesis of the KGD enzyme complex is repressed in some species and carbon flow through some CAC steps is reversed converting the CAC into a branched pathway that functions primarily with intermediary metabolism. The purple photo synthetic bacterium Rhodobacter capsulatus relies on the CAC for energy production under aerobic growth conditions on minimal media containing organic acids such as malate, succinate, and pyruvate. However, when growing under anaerobic photo synthetic conditions R. capsulatus produces energy by photosynthesis, although intermediates of the CAC are still required for the biosynthesis of bacteriochlorophyll and amino acids. It is believed that R. capsulatus does not operate an anaerobic branched version of the CAC, as in other facultative bacteria, but a less active cyclic one. Therefore, it is of great interest to understand the mechanism by which KGD enzyme activity is regulated and how this affects the operation of the CAC in R. capsulatus in response to fluctuating environmental conditions. I have isolated the R. capsulatus sucA gene encoding the E1o enzyme component of the KGD enzyme complex. Pulse labelling of R. capsulatus cultures growing aerobically and photosynthetically indicated that the sucA gene was transcribed at a higher level under aerobic growth conditions than under photosynthetic growth conditions. This increased level of transcription resulted in a 7-fold increase in the steady state levels of KGD mRNA, and a 9 to 13-fold increase in KGD enzyme specific activity in crude cell extracts under aerobic growth conditions, compared to anaerobic photosynthetic growth conditions. This regulation of the sucA gene in response to oxygen is different from any other R. capsulatus gene studied, and is probably part of a larger network of processes that takes place to regulate control of metabolic pathways in cells that shift between two environmental conditions.
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