Because of their photosynthetic lifestyle, plants have naturally developed elaborate mechanisms for controlling their responses to the quality and quantity of incident light. During periods of exposure to high intensity light (HL) the photosynthetic apparatus becomes over-excited and this excess energy can cause serious damage to the cell. Studies suggest that photosynthetic organisms may sense that they are receiving HL, and trigger the expression of factors that help the organism to acclimate to HL stress, by monitoring the redox state of the photosystem or through the activities of one or more blue/UV-A light (BL) photoreceptors. The long-term goal of this research is to identify and characterize the sensors and other regulatory elements that control gene expression in response to HL in cyanobacteria. Previous work has identified one such regulatory element. This factor, a histidine sensor kinase designated NblS, controls the expression of a number of important genes during HL stress. NblS is hypothesized to sense the redox stress on the photosystem during HL exposure and appears to also be involved in BL photoperception. An objective of this research is to explore the mechanism of HL sensing by NblS. The presence of a PAS domain within the sequence of NblS strongly suggests that it binds a redox-active cofactor. This possibility will be explored. The presence and nature of a cofactor bound to this domain will be determined by spectral analysis of the PAS region of NblS overexpressed in E. coli. Subsequent alignment-based, site-directed mutagenesis would be used to identify the residues important in binding of the cofactor. This information would serve as an important step towards understanding the sensory mechanism of NblS. Another objective of this research is to identify other regulatory factors involved in controlling gene expression in response to HL stress in cyanobacteria. This will be done by generation and analysis of additional mutants altered in expression of the HL- and BL-activated hliA gene. Mutants will be generated by transposon mutagenesis in the cyanobacterium Synechocystis PCC 6803. Combined use of the tagging mutagenesis method and knowledge of the entire genome sequence that is available for this strain will allow rapid identification of a number of factors involved in the HL-sensing process. It has long been recognized that numerous processes in all forms of life are moderated by light, and more recently it has become evident that a variety of systems can be controlled by intracellular redox or redox potential. The studies described here will provide greater understanding of how these signals are perceived and how they are used to control gene expression.
