We have used experimental and computational analysis of R. sphaeroides photosynthesis and other gene expression networks (Kaplan, Gomelsky, Donohue) (3-9, 12, 13, 15-18, 20). We have identified many new candidate photosynthesis genes with expression patterns that varied as a function of light intensity. Results from these experiments suggest there are many more light-regulated aspects of the photosynthetic lifestyle of this bacterium than previously appreciated. Ongoing genetic analysis confirms that mutations in some of these newly-identified photosynthesis block the ability of cells to use solar energy in the laboratory. We also carried out transcriptome and computational analysis of individual R. sphaeroides regulons. This identified additional genes that are directly regulated by individual transcription factors and refined the consensus sequence for master regulators of photosystem development. We also showed that PpsR indirectly regulates genes that do not contain the PpsR-binding sites, e.g. puf and puhA operons. This suggests that PpsR plays a more global role as a regulator of photosystem development than what was assumed before. A similar computational and microarray analysis of PrrA target genes has identified many new candidate promoters that are controlled by this master regulator of photosynthesis. We have begun bioinformatic, genetic and biochemical experiments aimed at elucidating more » the interactions of transcriptional pathways controlling photosystem development (PrrBA and AppA-PpsR). We carried out computational analysis designed to cluster oxygen-dependent genes in R. sphaeroides based on the transcriptome data for cells grown between 30% and 0% oxygen. As a result, new statistical tools for clustering expression profiles from DNA microarrays have been developed. We have analyzed the assembly, bioenergetic role and regulatory functions of the aerobic respiratory chain (Donohue, Edwards, Hosler, Kaplan)(10, 11, 14)(Hiser, 2000-4612). We have used computational, genetic and biochemical approaches to map the flow of electrons through the major bioenergetic pathways of this bacterium. From this, we have formulated and tested predictions for the major and minor routes of electron transport to the 5 predicted terminal oxidases; these are now being tested by genetic and physiological experiments. We have demonstrated a direct role for the R. sphaeroides cyt cbb{sub 3} terminal oxidase of the aerobic respiratory chain in controlling the activity of a master energy homeostasis pathway, PrrBA. This presents an opportunity for analyzing the ability of a cytochrome oxidase to act as a direct modulator of a second bioenergetic machine like the photosynthetic apparatus. Given the important bioenergetic and regulatory role of the aerobic respiratory chain, we have analyzed assembly of this machine. One of these proteins, Cox11p is a copper protein that plays a critical, yet poorly-understood, role cytochrome oxidase assembly in many bacteria and organelles. We have (for the first time) succeeded in expressing the soluble domain of Cox11p as a fusion protein with thioredoxin in E. coli and shown that the protein binds copper. This and a series of mutant Cox11p proteins are being used to analyze the role of copper in this protein and determine the role of Cox11p homologs in assembly of Cu-dependent bioenergetic machines. In collaboration with GTL researchers at Pacific Northwest National Laboratory, we analyzed and localized the R. sphaeroides proteome (Kaplan, Donohue, Smith)(1, 2, 19). We have determined the entire proteome of cells grown under different energy states and localizes what fraction of the proteome is contained in the major subcellular fractions of the cell, including the photosynthetic apparatus. This has identified many new and unknown proteins within this specialized solar energy harvesting machine that we would like to analyze with future DOE support. « less