Cyanobacteria are the simplest organisms known in which to explore the mechanisms of circadian biological clocks. We have identified a gene, cikA, which is integral to relaying environmental information to reset the phase of the circadian clock in Synechococcus sp. strain PCC 7942, the model system for prokaryotic circadian rhythm studies. With the goal of elucidating the molecular basis of the clock and its entrainment to the photic environment, we will exploit this discovery by determining whether CikA acts directly as a photoreceptor and by identifying its signaling partners and their biochemical activities. CikA is a member of the phytochrome family of proteins, but lacks the expected cysteine residue that provides a ligand for bilin chromophore attachment in phytochromes and some other phytochrome-like proteins. The C-terminal domain has a well-conserved histidine protein kinase motif, and a segment of similarity with the receiver domains of response regulator proteins of bacteria. We will purify CikA directly from the cyanobacterium to determine whether a chromophore is attached, and if so, its chemical identity and influence on CikA function. This will be facilitated by modifying the cikA gene to add an affinity tag to the protein, and confirming that the modified gene is functional through complementation of a cikA null mutant. The role of autophosphorylation of the histidine protein kinase domain in resetting will be assessed. Genetic screens, both by a yeast two-hybrid assay and transposon mutagenesis in the cyanobacterium, will be used to identify proteins with which CikA interacts. The roles of these CikA partners in the phase resetting input pathway will be determined as well. In addition to providing fundamental insights into this most basic of biological processes, the project is also likely to uncover functional properties of currently cryptic phytochrome homologs that are present in the genomes of diverse prokaryotes.
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