Related User Projects

• Structural Studies of a Family of Proteins from the Diurnal Cyanobacteria Cyanothece 51142 that Contain an Unusual Repeated Five-Residues Domain (RFR).
  Project Lead: Garry Buchko
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  As complete genome sequence information became available for many organisms, a novel family of proteins containing a tandem pentapeptide repeat was discovered that can be approximately described by the general sequence motif A[D/N]LXX. Today, the Pfam database lists 1461 pentapeptide repeat proteins (PRPs) (Pfam00805). In approximately two-thirds of these proteins the pentapeptide repeat is the only recognizable domain (Vetting, 2006). While the overwhelming majority of these proteins have been identified in prokaryotes, they are also found in eukaryotes, including one protein in humans. The number of chromosomal copies of PRPs is not evenly distributed in prokaryotic genomes. Photosynthetic cyanobacteria appear especially endowed with 16 PRPs identified in Synechocystis sp. strain PCC6803 and 40 in Nostoc punctiforme. The genome of the cyanobacterium Cyanothece sp. strain 51142 has recently been sequenced and 35 PRPs identified in its chromosome (Welsh and Pakrasi, personal communication). Cyanothece 51142 is a complex photosynthetic microorganism that sequesters carbon dioxide from the atmosphere. Understanding the biology and ecology of this organism is of central importance to DOE?s bioremediation efforts with our research supporting DOE?s science mission to build and operate ?state-of-the-art? scientific user facilities. Furthermore, understanding the functional and structural details of proteins from Cyanothece is part of a Membrane Biology Grand Challenge Project, lead by Dr. Pakrasi at Washington University in St. Louis. Towards fulfilling the goals of the DOE and the Membrane Biology Grand Challenge, we are characterizing the functional and structural properties of the PRP family of proteins that are especially prolific in cyanobacteria.
• Cyanobacteria Membrane Biology Grand Challenge: Systems Analysis of the Dynamics of Membrane Architecture, Composition, and Function- Proteomic, Metabolomic, and Metallomic Characterization
  Project Lead: Jon Jacobs
  Project Lead Institution: Pacific Northwest National Laboratory
  Abstract
  The strain Cyanothece sp. ATCC 51142, isolated from benthic waters off the coast near Galveston, Texas, is a unicellular strain approximately 4-7 μm in diameter along the major axis. This organism, which fixes nitrogen at very high rates, has developed a type of temporal regulation in which N2 fixation and photosynthesis occur at different times throughout a diurnal cycle, including very high levels of CO2 fixation and storage during light cycles for use in dark cycles. These aspects of carbon and nitrogen storage make Cyanothece almost unique among well-studied cyanobacteria and make it a key candidate for broad characterization. The analysis of gene products and metabolism in Cyanothece can provide tremendous information about how the photosynthetic unicell compensates for this complex multi-tasking effort. This project is directed towards developing approaches and methods for the rapid detection of large numbers of peptides and metabolites in an extremely broad manner that is highly sensitive (e.g., able to detect low level species), quantitative, and is facilitated by high-throughput measurements. In addition, we will develop procedures and techniques for global metallomic characterization of Synechocystis 6803 and Cyanothece 51142 organisms. In this project, we focus on developing methods based upon the use of high-resolution capillary liquid chromatography combined with either Fourier transform ion cyclotron resonance (FTICR) or high mass accuracy time-of flight (TOF) mass spectrometry (MS) to increase the comprehensiveness, quantitation and throughput of the proteomics and metabolomics analyses, and leverage the significant separations and mass spectrometry capabilities originally developed at PNNL in the Biological Systems Analysis and Mass Spectrometry group. We will apply various MS/MS methods in combination with accurate mass and LC elution time information to initially identify peptides and define ?tags? so that subsequent measurements can be made without the need for routine MS/MS analyses. A similar approach will be taken for cataloging and tracking, initially structurally-unidentified, metabolites. Using mass and time (MT) tags, broad proteome and metabolome measurements can be made with much higher throughput. Under the Specific Aims of the project we will: (1) develop approaches using pulse chase stable isotope labeling to quantitatively measure protein turnover rates and dynamics in time-course studies in proteomic methods using both Synechocystis 6803 and Cyanothece 51142 as model systems, (2) Develop procedures and techniques for tracking large numbers of metabolomic features from Synechocystis 6803 and Cyanothece 51142 in time-course studies encompassing the light and dark cycles. This includes improvements to existing software designed to deisotope and display mass spectral data, and development of novel software to address the specific challenges presented by global metabolomics datasets. In addition, we will begin to structurally characterize any features displaying interesting behavior (i.e. dramatic increase or decrease in concentration) through targeted MS/MS studies and other means, if necessary (eg. NMR, chemical derivatization).and (3) develop procedures and techniques for global metallomic characterization of Synechocystis 6803 and Cyanothece 51142 organisms.
• Electron microscopy imaging of cyanobacteria Synechcystis sp.
  Project Lead: Alice Dohnalkova
  Project Lead Institution: Environmental Molecular Sciences Laboratory
  Abstract
  As a part of Grand Challenges in Membrane biology, Synechocystis sp. will be investigated in regards of cell morphology and elemental analysis of minerals associated with their outer and inner membranes. We are investigating metabolic response of Synechocystis on increased levels of Mn, Fe, Zn and Cu in the environment, in correlation with the Circadian rhytm of periodical changing the light and darkness. Experiments will be done in 331 building, and cells will be brought to EMSL either fixed (SEM) or thin sectioned (TEM).
• Grand Challenge in Membrane Biology
  Project Lead: Himadri Pakrasi
  Project Lead Institution: Washington University in St. Louis
  Abstract
  In this grand challenge, we will use a systems approach to understand the network of genes and proteins that govern the structure and function of membranes and their components responsible for photosynthesis and nitrogen fixation in cyanobacteria (blue-green algae). A systems approach integrates all temporal information into a predictive, dynamic model to understand the function of a cell and the cellular membranes. These microorganisms make significant contributions to harvesting solar energy, planetary carbon sequestration, metal acquisition, and hydrogen production in marine and freshwater ecosystems. Cyanobacteria are also model microorganisms for studying the fixation of carbon dioxide through photosynthesis at the biomolecular level. The results of this Grand Challenge will provide the first comprehensive systems level understanding of how environmental conditions influence key carbon fixation processes at the gene-protein-organism level. Specific tasks associated with this grand challenge will be submitted individually.