Oxygenic photosynthetic organisms utilize high-energy electron transfer chains comprised of redox active intermediates and light harvesting complexes. While oxygen is a necessary byproduct of water oxidation and the source of photosynthetic electrons, its presence is also dangerous because leakage of electrons and excitation energy can interact with molecular oxygen to generate reactive oxygen species (ROS). Elaborate antioxidant networks and redox buffering systems have evolved to protect photosynthetic organisms from the threat of ROS. Glutathione (GSH) is a multifunctional molecule that is involved in core metabolism, detoxification of xenobiotics and in maintenance of cellular redox poise. The ubiquitous nature of glutathione and its importance to cellular metabolism has been observed in many organisms, however the specific roles of glutathione in photosynthetic organisms are not fully understood. To address these questions, we have generated several mutants in the glutathione biosynthesis and degradation pathways in the model organism Synechocystis sp. PCC 6803 (Synechocystis 6803), an oxygenic photosynthetic cyanobacterium.
We utilized targeted homologous recombination to generate deletion mutants of glutamate-cysteine ligase (GshA) and glutathione synthetase (GshB) in Synechocystis 6803. Our results indicate that GshA activity is essential for growth in cyanobacteria because we were unable to isolate a fully segregated Δ gshA deletion mutant. We did isolate a ΔgshB mutant strain that accumulates the biosynthetic intermediate γ-glutamylcysteine (γ-EC) instead of GSH. In this work, I have characterized the physiology of the ΔgshB mutant following environmental, genetic and redox perturbations.
The results presented here also shed light on the dynamic nature of the low-molecular weight thiol pool in cyanobacteria. We quantified the levels of cellular thiols in Synechocystis 6803 during exposure to multiple environmental and redox perturbations and found that conditions promoting increased cellular metabolism and increased ROS production, including during high-light treatment and photomixotrophic growth, lead to higher cellular thiol levels. Furthermore, the intracellular pools of thiols decrease when the cell exhibits reduced metabolic capacity during conditions such as nutrient deprivation and dark incubation. Sulfate limitation results in dramatically decreased cellular thiol contents in a short period of time. We found that the ΔgshB strain is sensitive to sulfate limitation and exhibits delayed recovery upon sulfate repletion, indicating that GSH is important for acclimation to sulfate limiting conditions. To facilitate our understanding of GSH degradation in Synechocystis 6803 during sulfate limitation, we generated a mutant lacking γ-glutamyltranspeptidase (Ggt), an enzyme with GSH degradation activity. However, the Δ ggt mutant still exhibited GSH degradation during sulfate depletion, indicating the presence of an alternative system or mechanism. We did find increased levels of GSH in the growth media of the Δggt strain compared to the WT, which suggests a role in GSH uptake or prevention of leakage.
Our results demonstrate that GSH is essential for protection from multiple environmental and redox perturbations in cyanobacteria. However, there are many pathways involved in maintenance of redox homeostasis in cyanobacteria. Therefore, we also aimed to determine whether these pathways function cooperatively to ameliorate damage from ROS. Several flavodiiron (Flv) proteins have been identified in Synechocystis 6803 that are involved in reduction of O2 to H2O without the formation of ROS intermediates. However, single Δflv3 mutants do not exhibit severe growth defects under normal conditions. Therefore, we generated a Δ gshB/Δflv3 mutant to examine whether these systems cooperate to maintain redox homeostasis. Our results show that the ΔgshB/Δflv3 mutant exhibits reduced growth than either of the single mutants when grown on solid media, suggesting a degree of interaction between these pathways in cyanobacteria.
|Advisor:||Pakrasi, Himadri B.|
|Commitee:||Blankenship, Robert E., Ho, Tuan-Hua David, Jez, Joseph M., Kranz, Robert G., Smith, Thomas J.|
|School:||Washington University in St. Louis|
|Department:||Biology & Biomedical Sciences (Plant Biology)|
|School Location:||United States -- Missouri|
|Source:||DAI-B 72/07, Dissertation Abstracts International|
|Keywords:||Cyanobacteria, Glutathione, Metals, Photosynthesis, Redox homeostasis, Ros|
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