Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Technology Development and Use
Proteomics and Metabolomics
112
Characterization of Rhodobacter sphaeroides by High Resolution Proteomic Measurements
Mary S. Lipton*1 (Mary.Lipton@pnl.gov), Timothy Donohue*2 (tdonohue@bact.wisc.edu), Samuel Kaplan*3 (Samuel.Kaplan@uth.tmc.edu), Stephen Callister1, Matthew E. Monroe1, Margie F. Romine1, Ruihua Fang1, Carrie D. Goddard1, Nikola Tolic1, Gordon A. Anderson1, Richard D. Smith1, Jim K. Fredrickson1, Miguel Dominguez2, Christine Tavano2, Xiaihua Zeng3, and Jung Hyeob Roh3
1Pacific Northwest National Laboratory, Richland, WA; 2University of Wisconsin, Madison, WI; and 3University of Texas Medical School, Houston, TX
Exploiting microbial function for purposes of bioremediation, energy production, carbon sequestration and other missions important to the DOE requires an in-depth and systems level understanding of the molecular components of the cell that confer its function. Inherent to developing this systems level understanding is the ability to acquire global quantitative measurements of the proteome (i.e. the proteins expressed in the cell). We have applied out state of the art proteomics technologies based upon high-resolution separations combined with Fourier transform ion cyclotron resonance mass spectrometry to obtain quantitative and high throughput global proteomic measurements of the photosynthetic bacterium Rhodobacter sphaeroides. Significant progress has been made addressing biological questions using high resolution proteomic measurements of cells, and fractions thereof, cultivated under varying conditions.
Rhodobacter sphaeroides 2.4.1 is α-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Aerobically it can grow by respiration as a chemoheterotroph. It can also be grown either photo- or chemo- lithotrophically on hydrogen and carbon dioxide. The organism can fix nitrogen under anaerobic conditions, and can use a wide diversity of terminal electron acceptors such as oxygen, metal oxides or oxyanions and an array of organic molecules as electron donors. When grown photosynthetically, it uses wavelengths of light in the near infra-red and contains a reaction center that is the ancestor of plant photosystem II. R. sphaeroides has been shown to possess two chromosomes, the larger of approximately 3.0 Mbp and the smaller of approximately 1.0 Mbp and 5 plasmids that together encode some 4600 gene products.
The initial mass tag database consisted of global proteomic preparations from the organism cultured under both steady state aerobic and photosynthetic conditions. However, important in the physiology of the organism is not just the global expression of proteins but also the localization of these proteins. For example, the transition of the organism between an aerobic to a photosynthetic state is accompanied by a synthesis of the photosynthetic membrane imbedded with the photosynthetic apparatus. It is therefore important to determine the localization of the proteins with in the organism to achieve a clear view of the physiology. To this end, cellular fractions of these organisms cultured under both highly aerobic conditions where photosynthetic membrane synthesis is repressed (30% O2) and photosynthetic cell states (low, 3W/m2, light intensity to maximize photosynthetic membrane synthesis) have been analyzed. Photosynthetic cells have been fractionated into 5 relatively discreet fractions (cytosol, periplasm, inner membrane, photosynthetic membrane and outer membrane) and the aerobic cells have been fractionated into 4 relatively discreet fractions (cytosol, periplasm, inner membrane, and outer membrane) in an effort to determine protein localization in the cell. We will report on the identification of ~XXXX total proteins from aerobic and photosynthetically-grown cells as well as the localization of proteins associated with assembly, function or control of the photosynthetic apparatus to individual subcellular fractions from steady-state photosynthetically grown cells.
The true understanding of the transition between the steady states will be achieved by a temporal study of the protein expression patterns in aerobically grown R. sphaeroides cells shifting to photosynthetic conditions. We have applied quantitative proteomics measurements to cells taken from a time course experiment of these cells transitioning between the two states. Preliminary studies are focused on the synthesis and deposition of the photosynthetic apparatus into the cells, however, through clustering analysis we will be able to identify other proteins that are important in this transition as well.
* Presenting author
