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Microbial Cell Project Abstracts

University of Wisconsin-Madison Department of Bacteriology, University of Delaware Department of Chemical Engineering, University of Wyoming Department of Molecular Biology, University of Mississippi Department of Biochemistry, and University of Texas Medical Center at Houston Department of Microbiology and Molecular Genetics

tdonohue@bact.wisc.edu

Our long-term goal is to understand and to capitalize on the metabolic activities of facultative microorganisms. To accomplish this, we will be acquiring a comprehensive understanding of metabolic pathways, bioenergetic processes, and genetic regulatory networks in the facultative phototroph Rhodobacter sphaeroides, strain 2.4.1. No single organism has the number of variety of metabolic abilities that are known to exist in this a-proteobacterium. The R. sphaeroides genome sequence predicts the existence of additional DOE-relevant pathways that were not thought to exist in this organism (http://genome.ornl.gov/microbial/ rsph/). The known metabolic potential of R. sphaeroides includes,

• photoheterotrophic growth with light in the absence of O₂ using a variety of organic carbon compounds as a source of carbon and reducing power,
• photoautotrophic growth with light in the absence of O₂ using CO₂ as sole carbon source and H₂ as a source of reducing power,
• chemoheterotrophic growth without light in the presence of O₂ using a variety of reduced organic compounds as a source of carbon and reducing power,
• 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
• fermentative growth without light in the absence of O₂.
• the ability to degrade organic or inorganic toxins (including heavy metal oxyanions and oxides).

In this project, the Microbial Cell Project (http://microbialcellproject.org/) is supporting a team of microbiologists, biochemists, and metabolic engineers to analyze and model the flux of metabolites, the components of bioenergetic pathways, the assembly of key bioenergetic complexes, and the linkages between energetic and global regulatory networks in this facultative bacterium. This poster will describe the major projects that have been initiated since this project began in September, 2001. It will provide an overview of efforts we have begun to

• analyze the 5 predicted terminal oxidases of R. sphaeroides. This work includes efforts to characterize the biochemical and bioenergetic features of each enzyme, to develop models that predict the flow of reducing power through each branch of the aerobic respiratory chain at different O₂ tensions, and to use a defined set of oxidase mutants to determine the contribution of each oxidase to aerobic energy generation.
• define the role of newly discovered components of the photosynthetic electron transport chain. This work includes the use of mutant strains and metabolic engineering principles to determine how reducing power from the cyt bc₁ complex is funneled to the photochemical reaction center, the cyt cbb₃ oxidase, or other electron acceptors.
• dissect the genetic regulatory networks that control the expression of key suites of genes. This work includes identifying the role of O₂-sensitive transcription factors like FnrL, signal transduction pathways such as PrrABC, or alternative sigma factors in regulating expression of electron carriers that are needed to generate energy, assimilate CO₂/N₂, or remove toxic compounds. Conditions are also being optimized for using DNA microarray technology to identify genes that are controlled by potential hierarchies of global regulatory networks.
• analyze the levels and turnover of metabolites that contribute to cellular energy generation in both steady state cultures and one that are adapting to new metabolic conditions. This work includes analysing those metabolites that set the energetic state or oxidation-reduction potential of the cell.
• analyze assembly of critical bioenergetic enzymes. This work includes using immunofluorescence microscopy to visualize the formation of bioenergetic machines like the photosynthetic apparatus in both wild type cells and mutants that lack presumed assembly factors.

¹Department of Plant Biology and the Photosynthesis Center, Arizona State University, Box 871601, Tempe, AZ 85287-1601
²Pasarow Mass Spectrometry Laboratory, Departments of Chemistry and Biochemistry, Psychiatry and Biobehavioral Sciences and The Neuropsychiatric Institute, UCLA, 405 Hilgard Avenue, Los Angeles, California 90095

wim@asu.edu

A completed genome sequence is only an excellent start: the next challenge is to determine the functional relevance of open reading frames for the physiology of the organism. To enhance the potential for functional analysis of open reading frames, a convenient transformation system should be available, and mutants impaired in key metabolic processes should be able to survive under specific conditions. These properties, and more, are found in the cyanobacterium Synechocystis sp. PCC 6803. This organism was the first phototroph of which a genomic sequence was determined, and it is spontaneously transformable, integrates DNA by double-homologous recombination, and can grow in the absence of photosynthesis or respiration.

Cyanobacteria are close to the ancestors of chloroplasts, but cyanobacteria maintain fully functional respiratory and photosynthetic complexes in the same internal membrane system, the thylakoids. Several redox-active components and proteins are shared between photosynthesis and respiration. A large number of Synechocystis mutants has been generated that are altered in one of the photosystems or in one or more of the respiratory complexes, and the properties of the various strains are being compared using a range of mostly in vivo techniques. These techniques include (1) redox measurements of the plastoquinone pool, (2) electron transport rate measurements using an oxygen electrode, (3) chlorophyll fluorescence analysis indicating the redox state of the system, (4) localization of protein complexes by means of fluorescent tagging, (5) ultrastructural analysis of membranes and cells, eventually leading to a 3-dimensional view of the cell, (6) quantitative analysis of metabolites such as organic acids using GC/MS, and (7) comparative proteomics of different mutants, the subject of the presentation by Whitelegge et al.

The Synechocystis Cell Project focusing initially on photosynthetic and respiratory processes has been started in September 2001, and builds on very significant insight that has been obtained since the genome sequence was completed. This insight includes, among others: (1) Cells can be grown in the absence of either oxygen evolution (by photosynthesis) or oxygen uptake (by respiration) when a fixed-carbon source is available; the identity of the electron acceptor in this case is being investigated. (2) Even though a 2-oxoglutarate dehydrogenase complex is missing according to the genome sequence, a complete TCA cycle has been demonstrated; an alternate shunt between 2-oxoglutarate and succinate is proposed. (3) A traditional membrane subunit of succinate dehydrogenase is missing according to the genome sequence; however, a very different subunit resembling that from Archaea is present and appears functional, suggesting a lateral gene transfer event. (4) The plastoquinone pool, a central redox buffer in both photosynthesis and respiration, is fairly reduced in darkness and is oxidized in the light; this is consistent with a much higher abundance of photosystem I (taking electrons from the pool) than photosystem II (donating electrons to the pool) in this cyanobacterium. (5) Pigment cofactors, including chlorophylls and carotenoids, can be altered somewhat in their chemical structure without major functional consequences; this illustrates the plasticity of pigment binding sites and of pigment function.

Together, the results obtained thus far on Synechocystis sp. PCC 6803 illustrate the major impact a genomic sequence can have on starting to understand the molecular physiology of a cell, particularly when the genomic sequence has been obtained on an organism that is easily accessible to targeted genetic modifications.

The Synechocystis Microbial Cell Project is funded by DOE (DE-FG03-01ER15251).

¹Bioinformatics Research Group, SRI International, 333 Ravenswood Ave, EK207, Menlo Park, CA 94025
²Stanford University

pkarp@ai.sri.com

We have used SRI’s Pathway Tools software to predict the metabolic-pathway complement of Caulobacter crescentus. The resulting prediction is stored within a pathway/genome database that integrates information about the genes, gene products, and biochemical pathways of Caulobacter. The poster will describe the computational method for predicting metabolic pathways, and the Pathway Tools components for querying and visualizing pathway/genome databases. The poster will also summarize what pathways were identified in Caulobacter.

Harley McAdams¹, Michael Laub², Peter Karp³, Lucy Shapiro¹, Alfred Spormann¹, and Charles Yanofsky¹

¹Stanford University
²Harvard University
³SRI International

mcadams@cmgm.stanford.edu

An interdisciplinary team from Stanford, Harvard, and SRI International is identifying and characterizing the complete genetic regulatory circuitry and metabolic pathways of the aquatic bacterium, Caulobacter crescentus. The global physiological responses of C. crescentus cells and cultures during starvation, during adaptation to exposure to toxic chemicals, during exposure to alternative, environmentally-relevant catabolic substrates, and in biofilms will be determined using DNA microarrays, metabolic biochemistry and metabolic profiling, pathway and circuitry modeling, and bioinformatics.

The initial step is to estimate the overall regulatory and metabolic networks in C. crescentus through gene expression microarray assays of the wild type strain and bioinformatics analysis. For example, gene expression of wild type cells will be assayed for a selected set of time courses when subjected to diverse environmental conditions, such as different nutrient levels, transition into and out of stationary state, sudden exposure to several stresses and to changed nutrients in the environment, and growth as biofilms. Cluster analysis and analysis of the timing patterns in these datasets will predict sets of genes that are regulated as cascades or cassettes. From sequence homologies and the temporal patterns, candidate regulatory genes will be identified. Then the Pathway Tools software will be used to identify C. crescentus metabolic pathways. Additional microarray studies as well as conventional genetic and biochemical analyses of both wild type and mutant strains will then be designed to verify the postulated regulatory network.

Initial results have identified the operon organization of the C. crescentus genome and time varying gene expression levels from synchronized cultures of both wild type and mutant cells. Also, tools to visualize and “browse” the genome structure have been constructed.

Alison Hottes, Swaine Chen, Lucy Shapiro, and Harley McAdams

Stanford University

ahottes@stanford.edu

With the increasing availability of both fully sequenced microbial genomes and microarray data, it is becoming easier to consider the overall organization of bacterial genomes. As part of the Stanford Microbial Cell Project, we have looked at two aspects of the aquatic bacterium Caulobacter crescentus’ genomic organization – its transcription unit (operon) structure and the distribution of GAnTC methylation sites within its genome.

1. The operon-level organization of a bacterial genome forms the foundation of that bacterial cell’s transcriptional regulatory network. We have used a probabilistic framework to predict the operon organization of the C. crescentus genome. Our computational method uses microarray data from both mutant and cell cycle studies, information about adjacent pairs of genes conserved across multiple bacterial genomes, and genomic spacing data. The algorithm makes extensive use of information about the size and spacing of transcription units in the more widely studied Escherichia coli.
2. In Caulobacter crescentus, CcrM is an essential cell cycle-regulated DNA methyltransferase that methylates the adenine in GAnTC sites. CcrM is not part of any known restriction/modification system. In the C. crescentus genome, GAnTC sites appear less frequently than would be expected by chance alone and are distributed non-uniformly around the chromosome. We present a variety of statistical studies, including correspondence analysis, that were conducted to determine a function for CcrM.

This research is part of the Stanford University Microbial Cell project. The objective of the project is to identify the complete transcriptional regulatory network of Caulobacter crescentus.

References

1. Craven M, Page D, Shavlik J, Bockhorst J, Glasner J. A probabilistic learning approach to whole-genome operon prediction. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2000; 8: 116-27.
2. Ermolaeva MD, White O, Salzberg SL. Prediction of operons in microbial genomes. Nucleic Acids Res. 2001 Mar 1; 29(5): 1216-21.
3. Karp, P.D., Riley, M., Saier, M., Paulsen, I.T., Paley, S. and Pellegrini-Toole, A. EcoCyc: Electronic Encyclopedia of E. coli Genes and Metabolism. Nucleic Acids Research, 28(1): 56 2000.
4. Salgado H, Moreno-Hagelsieb G, Smith TF, Collado-Vides J. Operons in Escherichia coli: genomic analyses and predictions. Proc Natl Acad Sci USA. 2000 Jun 6; 97(12): 6652-7.
5. Stephens C, Reisenauer A, Wright R, Shapiro L. A cell cycle-regulated bacterial DNA methyltransferase is essential for viability. Proc Natl. Acad. Sci. USA. 1996 Feb 6; 93(3): 1210-4.

Peter Agron and Gary Andersen

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551

agron1@llnl.gov

We have recently initiated a study aimed at taking advantage of the complete genome sequence of Caulobacter crescentus to study protein-protein interactions as a means to help elucidate genome function. As well as serving as a model system to study the cell cycle and cellular differentiation, this experimentally tractable gram-negative bacterium flourishes in aqueous environments with dilute nutrients, making it an attractive bioremediation vehicle. Our approach uses a commercially available two-hybrid system (Stratagene) in Escherichia coli. Vectors that encode fusions to two protein fragments (bacteriophage l cI and the N-terminus of the RNA polymerase a-subunit) confer a biological activity that can be easily assayed (penicillin resistance) if the two chimeric proteins bind. We plan to adapt this system to allow up to approximately 200 genes of interest to be tested for interactions using a random-fragment library. Here we present pilot studies to test the efficacy of the approach using genes known to encode interacting proteins. Interactions were tested for structural proteins involved in cell division (FtsZ/FtsZ, FtsZ/FtsA, Smc/Smc), regulatory proteins involved in chemotaxis (CheA/CheW and CheA/McpA), regulatory proteins involved in the cell cycle and differentiation (CtrA/DivK), and regulatory proteins involved in nutrient uptake (PhoR/PhoB). Several of these genes encode two-component histidine kinases and response regulators, an important class of signal transduction proteins that is very abundant in C. crescentus and crucial for many cellular processes. The results of the pilot studies and the outlook for large-scale experiments will be discussed.

Amudhan Venkateswaran¹, Marina Omelchenko¹, Hassan Brim¹, and Michael J. Daly¹

¹Uniformed Services University of the Health Sciences (USUHS) 4301 Jones Bridge Road Department of Pathology, Room B3153, Bethesda, MD 20814-4799
²National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894

Avenkateswaran@usuhs.mil; omelchen@ncbi.nlm.nih.gov

Bacteria belonging to the family Deinococcaceae are some of the most radiation resistant organisms discovered. Different species of the Deinococcus genus were examined for their metabolic diversity and resistance to oxidative stress and radiation. Of the known deinococcal species, the thermophilic Deinococcus geothermalis is the least resistant and the only one endowed with complete biosynthetic capabilities. By comparison, the other members of the genus show greater resistance, but are highly dependent on cysteine and nicotinic acid for growth, and secrete proteases. Following the recent sequencing and annotation of D. radiodurans, research on its resistance capabilities is expanding to include the consideration of global cellular processes within which protection and repair systems operate efficiently. We present data consistent with the hypothesis that metabolism of D. radiodurans has evolved to minimize production of oxygen free radicals. While defects in deinococcal metabolism severely limit growth in nutritionally restricted environments, they likely enhance recovery of cells with accumulated DNA damage.

Stephen Holbrook¹, Ursula Shulze-Gahmen¹, David Wemmer¹, James Berger¹, Sung-Hou Kim¹, Steven Brenner¹, and Michael Kennedy²

¹Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720
²Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352

srholbrook@lbl.gov

Deinococcus radiodurans (Dr) has the most efficient DNA repair and maintenance system of any organism yet identified. Insights into the general nature of DNA repair in all organisms may be achieved through detailed structural analysis of the proteins involved in the repair pathways of this organism. Current research is focused on two important components of the Dr DNA-repair system, the recFOR pathway that facilitates repair of double-strand DNA breaks by homologous recombination, and the Nudix family of nucleotide polyphosphate hydrolases, for which 21 distinct proteins are present in Dr, the most in any organism. Some members of the Nudix family limit mutations by hydrolyzing oxidized products of nucleotide metabolism that are mutagenic when incorporated into the genome.

The genes constituting the recFOR pathway have been successfully cloned in our lab from Dr as well as from the hyperthermophilic organism, Thermus thermophilus. Recombinant RecO and RecR are well-expressed soluble proteins while RecF tends to from inclusion bodies in bacterial overexpression systems. Purification and crystallization experiments of the soluble RecO/R proteins are under way and different expression systems for RecF will be tested in the future.

Bacterial clones for six recombinant Nudix proteins from Dr were obtained from M. Bessman (Johns Hopkins) and we have cloned three other Nudix proteins. All of the proteins are soluble and express well and they are currently being purified. One of the Nudix proteins (DR1025) was crystallized and X-ray diffraction data collected to high resolution (1.6Å). Crystallographic phasing and structure solution is under way. Another Nudix protein (DR0079) is under analysis by NMR methods.

¹Louisiana State University and A & M College, Baton Rouge, LA 70803 USA
²The Institute for Genomic Research, Rockville, MD 20850 USA

jbattis@lsu.edu

The D. radiodurans genome encodes essentially the entire ensemble of DNA repair proteins found in E. coli. With the exception of alkylation transfer and photoreactivation, all of the major prokaryotic DNA repair pathways are represented. This observation is significant, not because it says anything about why D. radiodurans is radioresistant, but because it confirms something long suspected; D. radiodurans possesses unique mechanisms for dealing with ionizing radiation-induced DNA damage. Clearly, the collection of identified repair proteins in D. radiodurans, in and of itself, is not sufficient to confer radioresistance. If it were, E. coli would be as radioresistant. Assuming that the identified repair proteins encoded by D. radiodurans perform the same functions as their E. coli homologues, it seems reasonable to expect that D. radiodurans expresses novel proteins that enhance this species survival. Of the 3187 open reading frames identified in D. radiodurans R1, only 1493 could be assigned a function based on similarity to other gene products found in the protein databases. Of the 1694 proteins of unknown function, 1002 are, at present, unique to D. radiodurans, showing no database match to any other previously sequenced gene. The secret to understanding the radioresistance of D. radiodurans is presumably found among these proteins of unknown function. To achieve the goal of defining the proteins necessary for the radioresistance of D. radiodurans, we have constructed a DNA microarray, and used this array to follow changes in gene expression as this species recovers from a sub-lethal dose (3000Gy) of ionizing radiation. Fewer than 50 loci showed significant increases in expression in response to this treatment during the first hour of recovery and approximately half of the induced genes encode proteins of unknown function. In a parallel study, R1 cultures were desiccated for a period of two weeks and gene expression followed during rehydration. The pattern of gene expression following desiccation was compared with that observed following ionizing radiation because desiccation, like ionizing radiation, induces DNA double strand breaks. The patterns of gene expression partially overlapped, identifying five loci (DR0003, DR0070, DR0326, DR0423, DRA0346) encoding hypothetical proteins. This result suggests that these loci are involved in D. radiodurans’ tolerance of DNA double strand breaks.

Department of Microbiology, University of Massachusetts, Amherst, MA 01003

dlovley@microbio.umass.edu

The long-term objective of this project, which has just begun, is to develop comprehensive conceptual and mathematical models of Geobacter physiology, and the interaction of Geobacter with its physical-chemical environment, in order to predictively model the behavior of Geobacter in a diversity of subsurface environments. Initial studies are focusing on Geobacter sulfurreducens because: 1) this microorganism has all of the unique physiological properties characteristic of Geobacter species; 2) the complete genome sequence is available; 3) a genetic system is available; 4) DNA-microarrays for the complete genome will be available; 5) it can be grown in chemostats; and 6) complementary functional genomic studies are underway.

In the first three years our objectives are to understand and model: 1) central metabolism in G. sulfurreducens; 2) electron transport to Fe(III) oxide, the electron acceptor supporting the growth of Geobacter in the subsurface; 3) electron transport to U(VI); 4) growth under the energy- and nutrient-limited conditions found in subsurface environments; 5) general regulatory mechanisms; and 6) response to environmental stresses such as oxygen and toxic metals. Chemostat studies are underway to collect physiological data to be compared with the predictions of the in silico model. Preliminary results on acetate uptake in the chemostats indicate that G. sulfurreducens can readily metabolize acetate at the low concentrations (ca. 10 µM) found in subsurface environments. Growth yields with fumarate as the electron acceptor are twice those with Fe(III) as the electron acceptor. This indicates that there may be an additional proton-translocation step in electron transport to fumarate. This hypothesis is being further evaluated.

Analysis of the genome predicted a number of previously unknown physiological characteristics which were evaluated in vivo. For example, putative chemotaxis genes were identified and subsequent investigations demonstrated that G. metallireducens was chemotactic toward Mn(II) and Fe(II), which are likely to guide the organism to Mn(IV) and Fe(III) oxides under anaerobic conditions. This is the first example of chemotaxis to metals. Although previous studies suggested that G. sulfurreducens and other Geobacter species are strict anaerobes, the genome contained genes coding not only for proteins involved in oxygen tolerance, such as catalase, superoxide dismutase, rubrerythrin, three potential rubredoxin genes, and a homolog of NADH/rubredoxin oxidoreductase, but also a homolog of a gene involved in oxygen reduction in Desulfovibrio spp., rubredoxin oxygen oxidoreductase.

Further physiological analysis demonstrated that G. sulfurreducens could recover from oxygen exposure for more than a day and we are currently investigating whether it is able to use oxygen as an electron acceptor in a manner similar to another “strict anaerobe”, Desulfovibrio. One of the more unexpected and fascinating stories of the G. sulfurreducens genome is the finding that G. sulfureducens’ metabolism is likely to be much more highly regulated than was hypothesized prior to sequencing the genome. Of particular interest are putative iron-sensing systems that are likely to be involved in controlling expression of key metabolic genes. The role of these and other putative regulator proteins are being intensively investigated.

In the future, the developing in silico model will be used to aid in the design of experimental approaches and as more information on gene function and regulation becomes available this will be incorporated into the developing in silico model. It is hypothesized that through this iterative approach it will be possible to evolve an in silico model that can predict the growth and activity of G. sulfurreducens in chemostats and, eventually, in the subsurface. In addition to making numerous contributions to the basic understanding of microbial physiology, a model capable of predicting the growth and activity of Geobacter species in the subsurface will greatly aid in the design of rational strategies for the in situ bioremediation of metal and organic contamination.

¹The Ohio State University
²University of British Columbia
³Univeresity of California at Los Angeles
⁴University of Iowa
⁵Pacific Northwest National Laboratory
⁶Oak Ridge National Laboratory

tabita.1@osu.edu

The objective of the Rhodopseudomonas palustris Microbial Cell Project is to examine how processes of global carbon sequestration (CO₂ fixation), energy generation from light, biofuel (H₂) production, plus organic carbon catabolism and aromatic hydrocarbon degradation and metal reduction operate in a single microbial cell. The recently sequenced Rhodopseudomonas palustris genome serves as the raw material for these studies. The control of all these processes appears to be integrated and the major aim of this study is to determine how this integrative metabolism is controlled and how certain aspects of metabolism, such as energy (H₂) production and carbon sequestration, might be enhanced in this versatile organism. We have assembled a team of investigators, from four academic and two DOE national laboratories, who share a common interest in bringing diverse approaches and types of expertise to bear on this important problem. Coordinated application of gene expression profiling, proteomics, carbon flux analysis and bioinformatics approaches are combined with traditional studies of mutants and physiological/biochemical characterization of cells. More specifically, functional analysis of the R. palustris proteome and global gene expression is considered within the context of the biochemistry and physiology of interactive control of energy generation and aerobic/anaerobic CO₂ assimilation and N₂ fixation, H₂ oxidation and H₂ evolution, and sulfur metabolism. These studies take advantage of the fact that R. palustris is phototrophic, can fix nitrogen, and may evolve copious quantities of hydrogen gas; the organism is unique in its ability to use a diversity of substrates for both autotrophic CO₂ fixation (i.e., H₂, H₂S, S₂O₃^2-, formate) and heterotrophic carbon metabolism (i.e., sugars, dicarboxylic acids, and aromatics, plus many others) under both aerobic and anaerobic conditions. As the project develops, intracellular localization and modeling of the expression of the key cellular processes will be undertaken.

¹Department of Microbiology, University of Iowa, Iowa City, IA
²Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN

caroline-harwood@uiowa.edu

Rhodopseudomonas palustris is a very successful photosynthetic bacterium that can be found in virtually any temperate soil or water sample on earth. It is among the most metabolically versatile of known bacteria and has many alternative ways of acquiring carbon and nitrogen and of generating energy. It can grow anaerobically in light, convert many diverse kinds of aromatic compounds to cell material, and convert gaseous nitrogen to ammonia. Each of these aspects of the biology of R. palustris is reflected in its 5.49 Mb genome and its 5,000 potential protein encoding genes. Moreover, R. palustris has apparently functionally redundant genes to encode each of these processes. As part of a project to use whole genome microarrays to define the genes and molecular regulatory mechanisms that are responsible for the metabolic versatility of R. palustris, we have begun construction of a pilot microarray. The microarray will be used to identify physiological conditions under which apparently functionally redundant genes involved in light harvesting, nitrogen fixation and aromatic compound/fatty acid degradation are differentially expressed relative to each other. The pilot microarray will also be used to identify regulatory genes that control the differential expression of each of these processes.

¹Pacific Northwest National Laboratory Richland, WA 99352
²Jet Propulsion Laboratory, Pasadena, CA
³University of Southern California, Los Angeles, CA
⁴Argonne National Laboratory, Argonne, IL

margie.romine@pnl.gov

We report on the use of coupled high resolution separation and high mass accuracy and sensitivity Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the protein content of outer membrane vesicles from the dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1. Outer membrane vesicles (MVs) are unique to Gram-negative bacteria, are initiated by the formation of “blebs” in the outer membrane, and are released from the cell surface during growth, trapping some of the underlying periplasmic contents in the process. Membrane vesicles provide an excellent means to identity proteins that are localized to the outer portions of the MR-1 cell envelope without disturbing cellular integrity or the need to further fractionate cells. Mass spectrometric analyses of vesicles isolated from MR-1 cells grown on LB supplemented with fumarate and lactate revealed the presence of 18 outer membrane and 12 periplasmic proteins. Proteins that were identified include electron transport pathway components (OmcA, OmcB, MtrB, CymA, fumarate reductase, and formate dehydrogenase alpha and Fe-S subunits), five putative porins, three proteases, proteins involved in protein maturation (PpiD and DsbA), and two transport proteins (long-chain fatty acids and tungstate). In addition, these samples contained FlaA flagellin proteins and the MshA pilin protein, head and tail proteins from prophage LambdaSo and MuSo2 which, along with several other putative inner membrane and cytoplasmic proteins, probably co-purified with vesicles. The presence of phage coat proteins in these samples suggests that a fraction of cells within MR-1 cultures are undergoing lysis during culture and may explain why proteins predicted to be associated with the inner membrane or cytoplasm were also detected in MV preparations. A comparison of the mass spectrometric results to heme stained gels suggests that at least two additional c-type cytochromes are present in the sample, one most likely being MtrA and the second a small c-type cytochrome under 14 Kd. We believe that these proteins were not detected by mass spectrometry because of the added mass of heme and are focusing efforts on methods to detect heme modified peptdies in MR-1 c-type cytochromes. The presence of electron transport proteins shown in vitro to be capable of reducing Fe(III) is consistent with related findings in S. putrefaciens CN32, a close relative of MR-1, where vesicles have been shown to mediate Fe(III), U(VI) and Tc(VII) reduction. Future directions will focus on defining the proteome of MR-1 MVs from cells cultured with different electron acceptors.

This work was supported by the Office of Biological and Environmental Research of the U.S. Department of Energy. Pacific Northwest National Laboratory is operated for the U.S. Department of Energy by Battelle Memorial Institute through Contract No. DE-AC06-76RLO

The Institute for Systems Biology, Seattle, WA

ekolker@systemsbiology.org

One of the major objectives of the DOE Microbial Cell Project [1] and more generally of the Genomes to Life Initiative [2] is “to develop the computational capabilities to integrate and understand genomic, proteomic, metabolic, regulatory, and physiology data and begin to model complex biological systems”. This necessitates combining the creativity of interdisciplinary teams incorporating complementary perspectives and principles from diverse fields and across wide strata of academic backgrounds. Work proposed by the Shewanella Federation [3] will integrate whole genome experimental approaches, including gene expression arrays and global protein expression studies, with comprehensive data analysis and modeling as well as biochemical, physiological, and genetic experiments. We will overview data analysis and integration issues associated with the Shewanella Federation. We will specifically focus on our recent advances in gene and protein expression studies of model microorganisms and how those advances will be integrated and applied to our study of Shewanella oneidensis.

References:

1. Drell, D., The DOE Microbial Cell Project: A 180 ° Paradigm Shift for Biology. OMICS: A Journal of Integrative Biology, 2001, 6(1), in press.

2. http://genomicsgtl.energy.gov/

3. http://shewanella.org/

This presentation describes a joint work with S. Stolyar, A. Keller, A. Nesvizhskii, D. Goodlett, E. Yi, S. Purvine (ISB), B. Tjaden, D. Haynor, A. Siegel (UW), A. Smith (UM), C. Rosenow (Affymetrix), E. Koonin (NCBI) as well as other members of the Shewanella Federation.

Correspondence to: ekolker@systemsbiology.org, 206.732.1278, fax 206.732.1260.

¹Oak Ridge National Laboratory
²Michigan State University
³University of Southern California
⁴Pacific Northwest National Laboratory
⁵Baylor College of Medicine
⁶University of California at San Diego
⁷Argonne National Laboratory

zhouj@ornl.gov

Large-scale sequencing of entire genomes represents a new age in biology, but the greatest challenge is to define gene functions and their regulatory networks at the whole-genome/proteome level. The key goal of this project is to explore whole-genome sequence information for understanding the genetic structure, function, regulatory networks and mechanisms of anaerobic energy metabolism in the metal-reducing bacterium, Shewanella oneidensis MR-1. Towards this goal, the following objectives will be achieved: (1) We will perform genome-wide mutagenesis using high-throughput random and targeted approaches to understand the functions of genomic sequences with emphasis on anaerobic energy metabolism and environmental responses. (2) We will dissect the regulatory networks and mechanisms of the proteins involved in anaerobic energy metabolism using integrated high-throughput genomic, proteomic and bioinformatic approaches by comparing gene expression patterns for both mutant and wild-type cells under different growth conditions. (3) We will define the functions of hypothetical proteins involved in anaerobic energy metabolism using integrated bioinformatic, genomic and proteomic approaches together with conventional biochemical methods. (4) Finally, we will simulate and predict the metabolic capabilities and cellular dynamics of S. oneidensis MR-1 in silico using constraints-based modeling approaches. Also, we will construct a central database to efficiently exchange and manage the massive data generated from this project. The proposed project will generate important information about the molecular mechanisms and regulatory networks of anaerobic energy metabolism and environmental responses. The genes identified in this study can be utilized as alternative molecular markers to measure activity and effectiveness of in situ bioremediation and will be valuable for the genetic engineering of bacteria for bioremediation purposes.

This research will be conducted as a collaborative project by scientists at Oak Ridge National Laboratory (ORNL), Michigan State University (MSU), University of Southern California (USC), Baylor College of Medicine (BCM), Pacific Northwest National Laboratory (PNNL), Argonne National Laboratory (ANL), and the University of California at San Diego (UCSD).