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Shewanella oneidensis MR-1 and related Shewanella sp.

Shewanella

Collaborators: Jim Fredrickson and Shewanella Federation PIs

The genus Shewanella is comprised of over 40 species and its members found in a wide range of environments worldwide including terrestrial and water saturated sediments, redox gradients in marine and freshwater environments, spoiled food, corroding oil pipelines, and the open ocean. More recently, metal-reducing Shewanella have been isolated from Columbia River sediments along the Hanford Reach including areas where metals and radionuclides from groundwater contaminant plumes are seeping into the river. Members of this group are best recognized for their versatility in respiratory metabolism, being able to gain energy for maintenance and growth through oxidation of organic compounds or H2 using compounds such as nitrate, nitrite, fumarate, trimethylamine oxide, thiosulfate, sulfur, and solid phase metals (e.g., Fe(III) or Mn(III, IV)) oxides as electron acceptors. Many Shewanella can also reduce relatively soluble and mobile contaminants including Tc(VII), U(VI), and Cr(VI) to lower oxidation states that are much less soluble and therefore less mobile in the environment. This respiratory versatility is thought to allow members of this genus to efficiently compete for resources in environments where electron acceptor type and concentration fluctuate in space and time.

Research Objectives:

  • Measure shifts in central metabolism that occur when MR-1 is grown under aerobic (20% DOT) vs. suboxic (0% DOT) conditions.
  • Identify proteins that localize to the cell surface when insoluble nutrients (e.g., chitin, starch) and/or electron acceptors (e.g., metal oxides, elemental sulfur) are provided for growth.
  • Characterize the role of iron homeostasis in supporting cell growth under suboxic and anaerobic conditions.
  • Determine the similarities and differences in growth strategies used by various species of Shewanella.

Rhodobacter sphaeroides 2.4.1

Rhodobacter

Collaborators: Profs. Tim Donohue (University of Wisconsin, Madison) and Sam Kaplan (U. Texas - Houston Medical School)

Rhodobacter sphaeroides generates energy by fermentation, respiration in the presence or absence of O2, or by a photosynthetic electron transport chain, that is the predecessor of the PSII of green plants, when light is present under anaerobic conditions. The R. sphaeroides photosynthetic apparatus is the best understood bioenergetic membrane system when one considers structural, functional and genetic information36. Biochemical, genetic and genome-enabled approaches are available to obtain a thorough, quantitative, understanding of the networks that impact solar-powered H2 production. The R. sphaeroides photosynthetic apparatus is a major source of the electrons for solar-powered H2 production. These electrons are released after light excites pigment-protein complexes within the intracytoplasmic membrane (ICM), a specialized domain of the inner membrane that is physically connected to, but functionally distinct from, the cytoplasmic membrane37, 38. The ICM is the functional equivalent of the plant chloroplast. It contains two light harvesting pigment-protein complexes that gather photons (B800-850, B875), a reaction center pigment-protein complex that reduces quinone upon oxidation by light energy, and enzymes to generate a protein gradient and synthesize ATP37.

In addition to the use of solar energy, R. sphaeroides is unique in its ability to oxidize a myriad of organic compounds (organic acids, sugars, methanol, various polyols, toxic compounds like formaldehyde), the use of compounds like sulfate, toxic metal oxides/oxyanions or thymine as electron acceptors and the ability to sequester CO2 even in the presence of fixed carbon sources. The metabolic potential extends to photoheterotrophic growth with light in the absence of O2, the photoautotrophic growth with light in the absence of O2 using CO2 as sole carbon source and H2 as a source of reducing power, the chemoheterotrophic growth without light in the presence of O2 using a variety of reduced organic compounds as a source of carbon and reducing power, the chemoautotrophic growth without light in the presence of O2 under using CO2 as sole carbon source and H2 as a source of reducing power, and the fermentative growth without light in the absence of O2. The research objectives in this section reflect ongoing work with R. sphaeroides where we have made significant progress in both the characterization of the organism and advancing the state of proteomics. Such experiments outlined below will advance the understanding of the organism, allow new understandings into energy production, guide new developments in the characterization of post-translational modifications and temporal studies, and help focus the needs for informatics and analytics in the "High-Throughput Proteomics Production Operations" project.

Research Objectives:

  • Identify proteins whose expression corresponds to changes in modes of energy production.
  • Determine the downstream effects on protein expression and modification that occur when genes that encode global regulators are deleted from the genome.
  • Measure the impact of singlet oxygen on protein expression and turn-over.

Geobacter species

Geobacter

Collaborator: Prof. Derek Lovley (University of Massachusetts)

The Gram-negative -proteobacterium Geobacter sulfurreducens is considered to be a representative of the Fe(III)-reducing Geobacteraceae that predominate in a diversity of subsurface environments where Fe(III) reduction is important. This organism is an anaerobic metal-reducing bacterium that can be used for bioremediation and electricity production by utilizing electrodes and electron transport on its outer surface. The physiology of the Geobacter species is of interest because they are frequently the most abundant microorganisms in soils and sediments in which microbial reduction of Fe(III) is an important process as well as the predominant organisms on electrodes harvesting electricity from a variety of sediments. Furthermore, Geobacter species have been found to be important agents for the bioremediation of groundwater contaminated with petroleum or toxic metals. The hallmark physiological characteristic of the Geobacter species is their ability to completely oxidize organic electron donors to carbon dioxide, transferring the electrons derived from organic matter oxidation onto extracellular electron acceptors such as Fe(III), toxic metals, humic substances, and electrodes.

The sequencing of the genome of G. sulfurreducens revealed many previously unknown physiological characteristics of this organism, such as the presence of 111 putative c-type cytochromes and a high proportion of proteins involved in environmental sensing. Furthermore, as in all microbial genomes, the G. sulfurreducens genome contains a large number of hypothetical genes encoding for putative proteins of unknown function. In order to better understand the physiology of G. sulfurreducens, it is important to know if these cytochromes and other predicted proteins are actually produced, and if so, under what conditions. Along with having considerable impact in the understanding of metal reduction and energy production, adaptive evolution studies will drive the production of visualization and informatics tools for data interpretation.

Research Objectives:

  • Identify spatial differences that occur in protein abundance in biofilms grown on fuel cell anodes.
  • Identify beneficial adaptations in cellular metabolism that lead to increased electricity production.
  • Identify proteins that are expressed under conditions that simulate those present at the Rifle IFC site.

Caulobacter crescentus

caulobacter

Collaborator: Prof. Lucy Shapiro (Stanford University)

C. crescentus is an excellent bacterial model for cell cycle processes typically attributed to eukaryotes and their multi-cellular development. C. crescentus asymmetrically divides into two different cell types, the swarmer cell and the stalked cell, which differ in morphology, behavior and differential programs of transcription and DNA replication. This dimorphic cell division is intrinsic to the cell cycle, and C. crescentus cell division always produces a nonreplicating swarmer cell and a replicating stalked cell. The swarmer cell differentiates into a stalked cell in order to replicate and divide. These cells possess distinct functional morphologies. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation. Protein phosphorylation is a key control mechanism of the cells progression through the cell cycle, and as such, this project drives the development of tools for the characterization of phosphorylation events in microbial systems.

Research Objectives:

  • Characterize the changes in the proteome content that corresponds with the progression through the cell cycle.
  • Characterize the cellular response to carbon and nitrogen starvation.
  • Monitor phosphorylation events that are associated with two component signal transduction.

Pelagibacter ubique and its meta-proteomic extension

Pelagibacter

Collaborators: Profs. Steve Giovannoni and Douglas Barofsky (Oregon State University)

SAR11, also known as Pelagibacter, is the dominant heterotrophic bacterial clade in the oceans. Roughly 25% of the 16S rRNA gene sequences retrieved from uncultured marine bacteria belong to the SAR11 group. Representatives of the SAR11 clade have been found in metagenome clone libraries from around the world. Investigations using fluorescence in situ hybridization (FISH) confirm that SAR11 bacteria often make up 25 to 35% of the total prokaryotic community in the surface waters of both coastal and open-ocean systems. The major breakthrough in the study of this clade of a-Proteobacteria was the first cultivation in 2002. Currently there are dozens of SAR11 isolates and two complete SAR11 genomes, with three more genome sequences in progress and others planned. Pelagibacter has the smallest genome found in free-living cells - 1,308,759 bp. Cultivation in natural seawater continues to be the only means of growing SAR11 isolates in laboratories. One of the first major metagenome sequencing studies published gave testimony to the environmental significance of the SAR11 clade - it accounted for a quarter of all of the 16S rRNA genes reported by Venter et al. in their study of the Sargasso Sea microbial meta-genome. Recent advances in proteomics sensitivity and the ability to analyze small samples have enabled this collaboration and this project has significant implications in the developing area of meta-proteomics. The advantage of the genome sequence of the SAR 11 organism and the genetic diversity represented in the Venter database will not only provide significant insights into the biological evolution of organisms with in a community, but will also provide a rich test bed for the advanced analytical and informatics tools being developed in "High-Throughput Proteomics Production Operations" project.

Research Objectives:

  • Identify mechanisms by which Pelagibacter survive during stationary phase growth.
  • Characterize differences in the stationary phase proteome composition of Pelagibacter that occur when N, P, Fe, or energy (carbon) was the limiting nutrient that caused the cell to enter stationary phase.
  • Characterize the regulatory elements that effect nutrient limitation.
  • Characterize the growth status of natural Pelagibacter populations and the factors controlling their growth in nature.

Poplar

Poplar

Collaborator: Dr. Jerry Tuskan (Oak Ridge National Laboratory)

Populus is the fastest growing tree species in North America and has been identified as a potentially important crop species for converting plant biomass to liquid fuels. Populus species are broadly adapted to nearly all regions of the U.S., and hybrid clones have demonstrated 10 dry tons per acre productivity on a commercial scale. Still, improvements in growth rate, cell wall composition, drought tolerance, and pest resistance are required before this species reaches it potential as an energy crop. Traditional genetic improvement in all of these traits is possible, but the timeframe for relevance to DOE mission dictates that the domestication process be accelerated. Recently the genome of Populus has been sequenced and annotated by a large international consortium lead by the Joint Genome Institute and Oak Ridge National Laboratory. The ab initio annotation indicates that there are between 30 and 45 thousand genes in the Populus genome, with over half of these falling into the hypothetical category. Modern genetics and genomics techniques can be applied to Populus to accelerate domestication, but first predicted gene models need to be verified. Global characterization of tissue specific proteins would advance this cause and would facilitate the development of a dedicated crop for conversion to liquid fuel.

Poplar represents a prototypical subject for proteomic characterization of more complex systems. Along with creating the necessary mass and time tag database to enable subsequent high-throughput sample analyses, we will utilize data to improve the genome annotation, identify protein splice variants, and quantitative measurements of different tissue types or affinity purified ubiquinated proteins extracted from both Poplar and Arabidopsis tissues. This project serves as the pilot study to characterize the different tissues in plants (e.g., roots, leaves, and stems) as well as tissues at a finer granularity, and as such, the methods developed in these studies will be applicable to other plant systems. The extension of such studies to other plants will be dictated by the needs of the Genomic Science Program Bioenergy Research Centers that are anticipated to have a significant science focus on plant biology.

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BER-PNNL Proteomics