The long-term objectives of this research program are to develop a detailed understanding of the structure, function, biogenesis, and regulation of the cyanobacterial photosynthetic apparatus. The dual goals of providing a detailed understanding of how light-harvesting anennae function as well as providing molecular tools which are especially important in characterizing gene regulation responses of cyanobacteria to nutrients and light intensity are being pursued. The RNA polymerase of Synechococcus sp. PCC 7002 apparently employs several different sigma factors. The hypothesis that global patterns of gene expression in cyanobacteria are controlled by promoter selection by these multiple sigma factors will be tested. Sigma factor proteins will be overproduced in E. coli and reconstituted with core RNA polymerase for in vitro transcription studies. The long-term aim is to correlate sigma factors with specific promoter sequences and with patterns of gene expression. All oxygen- evolving photosynthetic organisms exhibit an adaptive phenomenon called """"""""state transitions"""""""" by which light energy is distributed between the two photosystems to maximize the overall rate of photosynthetic election transport. The ApcD gene product is required for state transitions to occur and for energy to be transferred to Photosystem I. The hypothesis that post-translational modification of ApcD controls light energy distribution within the phycobilisome will be tested. Additional experiments will seek to demonstrate the contact partner of ApcD on the Photosystem I reaction center. Strains, plasmids, and methods to create site-specifically altered phycocyanins have been generated. These tools will now be used to address specific aspects of structure and function for phycocyanin through site-directed mutagenesis. Spectroscopic analyses of the mutant phycocyanins will be performed, and proteins exhibiting interesting defects in light-energy harvesting will be studied in collaboration with Dr. Robert Huber by X-ray crystallography. The ApcE protein plays critical roles in energy transfer to Photosystem II as well as in directing core assembly in the phycobilisome. Additional structure and function studies will be performed on this interesting protein, and the hypothesis that phycobilisome core structure is intrinsically determined by the number of linker domains in ApcE will be tested. The proposal that phycobilisomes of Anabaena sp. PCC 7120 contain four classes of peripheral rods and a total of eight rods attached to their cores will also be tested. The location of ferredoxin NADP+ oxidoreductase in phycobilisomes will be studied by immuno-electron microscopy, and the role of this molecule in phycobilisomes will studied by construction of a mutant lacking the recently identified CpcD-like domain. Finally, a bank of interposon- or transposon-tagged mutants will be created which will be screened for mutations affecting phycocyanobilin synthesis, covalent attachment of phycocyanobilin to phycobiliproteins, and state transitions. The successful completion of this program should significantly extend knowledge of mechanisms regulating gene expression in the photoautotrophic cyanobacteria and will additionally provide significant new insights into how light energy is efficiently harvested and distributed to the photosystems.
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