Abstract
The cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803) is a model organism used extensively to study photosynthesis and environmental stress responses. Its ability to integrate foreign DNA into its genome makes it a valuable organism for genetic engineering. Similar to many other cyanobacterial species, Synechocystis 6803 is able to grow photoautotrophically at neutral to alkaline pH ranges. Interestingly, strains carrying mutations in the PS II extrinsic proteins display a pH-sensitive phenotype where strains grow photoautotropically at pH 10.0 but growth is inhibited at pH 7.5. This project focuses on the pH-sensitive mutant ΔPsbV:ΔCyanoQ. Constant exposure of the ΔPsbV:ΔCyanoQ mutant to pH 7.5 has resulted in a response that restored photoautotrophic growth of two ΔPsbV:ΔCyanoQ mutant cultures. These pseudorevertant strains have switched from the mutated trait to the original phenotype. The two pseudorevertants were isolated: ΔPsbV:ΔCyanoQrev1 and ΔPsbV:ΔCyanoQrev2. Characterisation of the two pseudorevertants, revealed that the ΔPsbV:ΔCyanoQrev1 pseudorevertant, is able to assemble PS II and evolve oxygen. The ΔPsbV:ΔCyanoQrev2 pseudorevertant, can also assemble PS II, evolve oxygen and transfer electrons from PS II to QA and QB. To gain an understanding of what has occurred within the two pseudorevertant strains to allow growth in BG-11 buffered at pH 7.5, whole genome sequencing was conducted to isolate candidate genes that could be responsible for growth. Although, there were no candidate genes obtained for the ΔPsbV:ΔCyanoQrev1 pseudorevertant, other possible factors were considered such as the expression of known stress-response genes and sigma factors that could contribute to the growth of the ΔPsbV:ΔCyanoQrev1. In contrast, the candidate genes that were isolated from the ΔPsbV:ΔCyanoQrev2 pseudorevertant are thioredoxin C (trxC) and Photosystem II (PS II) manganese stabilizing protein (psbO). The trxC gene does not have a well-established function in Synechocystis 6803; however, thioredoxins are important for the regulation of proteins and enzymatic pathways. On the other hand, the psbO gene stabilizes the manganese cluster for oxygen evolution. This study indicates that both the trxC and psbO are two potential genes that could be involved in growth restoration of pH-sensitive strains in pH 7.5.