115. The Comprehensive Microbial Resource

Owen White, Jeremy Peterson, Jonathan A. Eisen, and Steven L. Salzberg

The Institute for Genomic Research, Rockville, MD 20850

owhite@tigr.org

One of the challenges presented by large-scale genome sequencing efforts is the effective display of information in a format that is accessible to the laboratory scientist. Conventional databases offer the scientist the means to search for a particular gene, sequence, or organism, but do little in the way of dispaying the vast amounts of curated information that are becoming available. TIGR has developed methods to effectively "slice" the vast amounts of data in the sequencing databases in a wide variety of ways, allowing the user to formulate queries that search for specific genes as well as to investigate broader topics, such as genes that might serve as vaccine and drug targets.

The Comprehensive Microbial Resource (CMR) is a facility for annotation of TIGR genome sequencing projects, and a web presentation of all of the fully sequenced microbial genomes, the curation from the original sequencing centers, and further curation from TIGR (for those genomes sequenced outside TIGR). The web presentation of the CMR includes the comprehensive collection of bacterial genome sequences, curated information, and related informatics methodologies. The scientist can view genes within a genome and can also link across to related genes in other genomes. The effect is to be able to construct queries that include sequence searches, isoelectric point, GC-content, GC-skew, functional role assignments, growth conditions, environment and other questions, and isolate the genes of interest. The database contains extensive curated data as well as pre-run homology searches to facilitate data mining. The interface allows the display of the results in numerous formats that will help the user ask more accurate questions. This resource should be of value to the scientific community to design experiments and spur further research. Resources of this type are an essential tool to make sense of bacterial genome information as the number of completed genomes continues to grow.

116. The Pseudomonas putida KT2440 Genome Sequencing Project

Karen E. Nelson, Hoda Khouri, Erik Holtzapple, Jeff Buchoff, Michael Rizzo, Azita Moazzez, Kelly Moffat, Kevin Tran, Hean Koo, P. Chris Lee, Daniel Kosack, Bradley Slaven, Helmut Hilbert, Burkhard Tuemmler, and C.M. Fraser

The Institute for Genomic Research, Rockville, MD 20850

kenelson@tigr.org

Pseudomonas putida is a ubiquitous soil bacterium that has significant potential for bioremediation of numerous compounds. To determine the complete genome sequence of strain KT2440, a genome sequencing project was initiated in January 1999 as a collaboration between The Institute for Genomic Research in Rockville MD, and a German Consortium (see http://www.tigr.org/tdb/mdb/mdb.html), with primary funding from the Department of Energy. The 6.1 Mbp genome is being sequenced by the random shotgun method, and multiple sized large insert libraries (10 kb, 40 kb) are acting as a scaffold for the genome. At the end of random sequencing, there were a total of 392 sequencing gaps, many of which have been closed by editing off the ends of assemblies, or by sequencing clones that span the respective gaps. The high GC content of the genome is highlighted in long stretches of G's and C's encountered in both sequencing and physical gaps, and through which we have had problems sequencing by traditional methods. Sequencing of short reads that point into gaps, dye primer chemistry, transposon mutagenesis on selected spanning clones, as well as ET chemistry were used for most of the difficult areas. Multiplex PCR and micro-library construction, have also assisted in resolving and ordering the RNA operons. Grouper and autoprimer (TIGR softwares) were improved or modified to deal with the size of the genome. Ultimately, the P. putida genome sequence will identify the real potential of this organism in various biotechnological areas including the production of natural compounds, and remediation of polluted habitats.

117. The Genome of Geobacter sulfurreducens

B.A. Methe¹, L. Banerjei², W.C. Nierman², O. Snoeyenbos-West¹, S. Sciufo¹, and D.E. Lovley¹

¹University of Massachusetts, Amherst, MA 01003 and ²The Institute for Genome Research, Rockville, MD 20850

bmethe@microbio.umass.edu

The complete genome sequence of Geobacter sulfurreducens is currently being determined to better understand its genetic potential. G. sulfurreducens is an important member of a family (Geobacteraceae) of delta Proteobacteria capable of oxidizing organic compounds including aromatic hydrocarbons to carbon dioxide with Fe(III) or other metals and metalloids including U(VI), Tc(VII), Co(III), Cr(IV), Au(III), Hg(II), As(V) and Se(VII) serving as the terminal electron acceptor. It is the dominant group of iron reducing microorganisms recovered from a wide variety of aquifer and subsurface environments when both molecular and traditional culturing techniques are used. Geobacter plays a critical role in the biogeochemical cycling of carbon, iron and other metals. Its genetics and physiology are a subject of intense study in part due to the importance that these processes can play in the remediation of contaminated anaerobic subsurface environments. The determination of the G. sulfurreducens genome is being accomplished using a random shotgun cloning approach to provide at least six-fold coverage of a 1mb genome followed by closure of remaining physical or sequence gaps. TIGR Assembler software and other computer programs developed by The Institute for Genome Research are used to assemble the genome and aid in gap closing, finishing and annotation. Searches of sequences and contigs from the early random phase of sequencing using the BLAST algorithm and database have produced high scores with low expect values indicating significant homologies to proteins contained in the database. These include enzymes considered important to basic housekeeping functions such as tRNA synthases and amino acid synthesis as well as those essential to other metabolic processes known to occur in G. sulfurreducens including nitrogen fixation. A number of sequences have produced no significant alignments indicating the likelihood of genes encoding for novel functions. Of further significance has been the extension of N-terminal sequences previously obtained from cytochromes known to be important in dissimilatory iron reduction. Thus, the genome will provide information crucial to the further understanding of this important metabolic process.

118. The Haloferax volcanii Genome Project

Rajendra J. Redkar, Joe J. Shaw, Gary G. Bolus, Mary Lee Ferguson, Troy A. Horn, and Vito G. Delvecchio

Institute of Molecular Biology and Medicine, University of Scranton, Corner of Monroe and Ridge Row, Scranton, PA 18510

Vimbm@aol.com

Haloferax volcanii is a salt-loving archaeon belonging to the family Halobacteriaceae. Halophilic archaea exhibit obligate halophilism and require 2-5 M salt concentration for viability. At lower concentrations of salt (about 1 M), cells become distorted, leading to cell lysis and death. The cytoplasm of halophiles contains very high internal concentrations of K+ and Na+, and is iso-osmotic with the environment. These bacteria have evolved metabolic and synthetic machinery that functions at high concentrations of salts, concentrations that are typically lethal for other organisms. Future research on halophilic archaea will provide better insight into early evolution of microorganisms and fundamental knowledge of biochemical and genetic events in organisms living in extreme environments.

H. volcanii cells are disk shaped and show involuted forms in the presence of NaCl. H. volcanii is a chemoorganotroph requiring complex nutrient medium and 1.5-2.5 M NaCl for growth. Cultures may be grown in the laboratory at 37°C with gentle shaking, however better growth is achieved at 42°C. In the laboratory, H. volcanii produces a characteristic pink pigment. Overall, the organism can be easily cultivated and maintained in the laboratory. Moreover, auxotrophic mutants are available, the cells are easily transformable, and genetic manipulation systems such as shuttle vectors and expression vectors are available for functional studies.

The genome size of the H. volcanii is estimated to be ~4.2 Mb. About 90% of the genome has 65% (G + C) content while remaining genome has 55% (G + C) content. The genome is composed of a chromosome (2,920 kb) and 4 plasmids, viz. pHV1 (86 kb), pHV2 (6.4 kb), pHV3 (442 kb) and pHV4 (690 kb). Plasmid pHV3 was selected for the first phase of sequencing operation. Sixteen overlapping cosmids were supplied by our collaborator Dr. Robert Charlebois, University of Ottawa, Ottawa, Canada, and used to make individual shotgun libraries. The average size of inserts in these randomly fragmented libraries is 2-3 kb. The sequencing reactions were performed on both ends of the clones using Big Dye Terminator chemistry to achieve 6-8X coverage. The data has been collated into several large assemblies using different software packages and the gaps in the assemblies are being located for closure. The annotation of data has started simultaneously to identify genes on pHV3. A progress report and the future plans on H. volcanii sequencing project will be presented.

119. The Caulobacter crescentus Genome Sequencing Project

Tamara Feldblyum¹, William C. Nierman¹, Nikhil Phadke², Peter Ulintz², and Janine Maddox²

¹The Institute for Genomic Research, Rockville, MD and ²Department of Biology, University of Michigan

tamaraf@tigr.org

Caulobacter crescentus is a member of the alpha subclass of the proteobacteria which also include Rickettsia, Rhizobium, Agrobacterium and Brucella species. It is the most prevalent non-pathogenic bacterium in nutrient-poor fresh water streams and is also found in marine environments. It is one of the organisms responsible for sewage treatment. Caulobacters are being modified for use as a bioremediation agent for removing heavy metals from wastewater.

Caulobacter crescentus has been extensively studied because it exhibits a well-defined developmental pattern that is independent of environmental stress. The free-swimming morphologically distinct swarmer cell progresses to an anchored stalked cell, the only cell type capable of genome replication and cell division. Cell division of the stalked cell splits out a swarmer daughter cell.

C. crescentus has a genome size of 4 Mb, with G+C content of about 66.5%. Tremendous power for genome assembly was brought to this project through the use of a 2 and 10 kb insert size 2 plasmid library strategy. In sequencing this organism at TIGR, 65,588 random sequence reads from both ends of plasmid clones were used to assemble the genome into only five groups comprising essentially all of the genome sequence. A preliminary review of C. crescentus ORFs revealed by the sequence is provided.

120. Unusual Features of Radioresistant Bacterium Deinococcus radiodurans Genome Revealed by Comparative-Genomic Analysis

Kira S. Makarova^1,2, L. Aravind², Roman L. Tatusov², Eugene V. Koonin², and Michael J. Daly¹

¹Uniformed Services University of the Health Sciences, Bethesda, MD 20814-479 and ²The National Center for Biotechnology Information, The National Institutes of Health, Bethesda, MD 20814

makarova@ncbi.nlm.nih.gov

In-depth analysis of Deinococcus radiodurans genome reveals some unusual features, which may be relevant to its extreme radioresistance and desiccation resistance. Comparison of the Deinococcus gene products to the collection of Clusters of Orthologous Groups of proteins (COGs) allowed us to identify not only a set of genes which are shared with all or most bacteria, but some surprisingly missing genes, including those for several enzymes involved in repair and recombination. Using this information, we tentatively reconstructed the metabolic pathways, repair and recombination systems and stress response mechanisms of D. radiodurans. The comparative analysis helped in identifying phylogenetic affiliations of Deinococcus and sets of genes with unusual phylogenetic patterns, especially those that are shared with thermophilic archaea and bacteria, indicating a possible thermophilic ancestor for Deinococcus. We described several protein families that are specifically expanded in the genome of D. radiodurans, namely possible nuclease inhibitors, specific transcriptional regulators and desiccation-related proteins, which could contribute to the radioresistance and desiccation resistance of this bacterium. Some additional unique multidomain proteins, which could be involved in novel repair or stress-response-related mechanisms were detected. Investigation of short repeats in Deinococcus resulted in the identification of their mosaic nature and suggested that they could contribute to the recombinational proficiency of this organism.

121. Protein Expression in Methanococccus jannaschii and Pyrococcus furiosus

C.S. Giometti¹, S.L. Tollaksen¹, H. Lim², J. Yates², J. Holden³, A. Lal Menon³, G. Schut³, M.W.W. Adams³, C. Reich⁴, and G. Olsen⁴

¹Argonne National Laboratory, Argonne, IL; ²University of Washington, Seattle, WA; ³University of Georgia, Athens, GA; and ⁴University of Illinois, Urbana, IL

csgiometti@anl.gov

Complete genome sequences are now available for both Methanococcus jannaschii and Pyrococcus furiosus. The open reading frame (ORF) sequences from these completed genomes can be used to predict the proteins synthesized, but laboratory methods are needed to verify those predictions. Two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry of peptides isolated from the gels is being used to determine the constitutive expression of proteins from these two Archaea and to explore the regulation of expression of non-constitutive proteins. The most abundant proteins (i.e., those easily detectable by staining with Coomassie Blue R250) from cells grown in minimal nutrient media have been isolated and analyzed. Using a combination of matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry, 100 proteins expressed by M. jannaschii and 50 proteins expressed by P. furiosus have been related to specific ORFs in the respective genome sequences. The molecular weights and isoelectric points determined by the positions of proteins in the 2DE patterns are compared with the ORF-predicted molecular weights and isoelectric points for each microbe. Numerous instances of multiple proteins with different molecular weights or isoelectric points being associated with the same ORF have been observed. Possible reasons for such multiplicity include the incomplete unfolding of these highly stable proteins prior to electrophoresis, the non-dissociation of subunits, post-translational modifications such as phosphorylation (multiple proteins with the same identity but different isoelectric points) or peptide cleavage (multiple proteins with the same identity but different molecular weights). Preliminary experiments to change the protein expression of these organisms by altering growth conditions have revealed significant quantitative changes in a small number of the proteins visible in 2DE patterns. Correlation of proteins expressed with specific ORFs is now focused on those proteins showing quantitative changes in expression and on less abundant proteins. The observed protein abundances and changes in abundance from these proteomic studies could be useful for validation of predictions of protein expression based on ORFs.

This work is supported under Contract No.W-31-109-ENG-38 with the U.S. Department of Energy.

122. Detection of Non-Cultured Bacterial Divisions in Environmental Samples using 16S rRNA-Based Fluorescent in situ Hybridization

Cheryl R. Kuske, Susan M. Barns, and Stephan Burde

Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545

Kuske@lanl.gov

Microbial genome sequencing projects have focused primarily on species that can be easily cultured. However, readily cultured bacteria are only a small fraction of the total bacterial diversity present in the environment. Diverse bacteria representing novel divisions have been identified in many natural environments using 16S rDNA sequence analysis. Microbial processes in these environments are of critical importance to the biosphere and the non-cultured bacteria residing there are a valuable resource for novel genomic information. We have identified novel bacterial divisions from 16S ribosomal RNA gene libraries generated from DNA of a volcanic cinder field and an arid sandstone soil. Using RFLP and sequence analysis, we have analyzed 800 bacterial rDNA sequences obtained from the two arid environments. The majority of sequences were members of recently identified bacterial divisions that have no, or very few, cultivated members (Kuske et al. 1997. AEM 63:3614-3621, Hugenholtz et al. 1998 J.Bact. 180:366-376). Using PCR primers specific for two of these divisions, Acidobacterium and OP11, and their subgroups, we have detected both divisions in local hot/warm spring microbial mats and sediments. Analysis of cell abundance of members of these groups is under investigation using fluorescently labeled rRNA probes and fluorescence microscopy. We plan to collect bacterial cells directly from the environmental samples using flow cytometry and cell sorting. The pooled DNA of non-cultured bacteria will be a valuable resource of genetic material for comparative analyses of conserved and novel gene families, and for targeted genome sequencing.

123. Diversity of Metal Reducing Bacteria from Ecological, Physiological and Genomic Perspectives

Jizhong Zhou^1,2, Guangshan Li¹, Alison Murray², Yul Roh¹, Heshu Huang¹, Ray Stapleton¹, Qiaoyun Qiu², John Heidelberg³, Claire Fraser³, Douglas Lies⁴, Kenneth H. Nealson⁴, and James M. Tiedje²

¹Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; ²Center for Microbial Ecology, Michigan State University, East Lansing, MI 48824; ³The Institute for Genomic Research, Rockville, MD 20850; and ⁴Department of Geology and Planetary Sciences, Jet Propulsion Laboratory and California Institute of Technology, Pasadena, CA 91109

zhouj@ornl.gov

Microbial metal reduction plays an important role in biogeochemical cycling of carbon and nitrogen as well as in bioremediation of metals, radionuclides and organic contaminants. To further investigate their diversity, metal-reducing bacteria were isolated from a variety of extreme environments including the deep terrestrial subsurface, Siberia and Alaska permafrost soils, continental margin marine sediments and Hawaii deep-sea water. Thermophilic isolates from terrestrial subsurface formations that had been geologically and hydrologically isolated for about 200 million years were able to use glucose, pyruvate, lactate, acetate and hydrogen as electron donors, and were able to reduce iron, manganese, chromium and uranium, as well as produce magnetite at 50-75 °C. The psychrotrophic isolates were able to use iron, manganese, and cobalt as electron acceptors, and were able to produce magnetite at 0 °C. A few isolates were able to reduce cobalt at - 4 °C and produce siderite using CO₂. Phylogenetic analyses indicated that the thermophilic iron-reducing bacteria were closely related to Thermoanaerobacter ethanolicus whereas the psychrotrophic iron-reducing bacteria were related to the members of the Shewanella genus. Although the psychrotrophic metal-reducing bacteria were able to use nitrate as electron acceptor, physiological studies and the comparisons of whole genome sequences from Shewanella oneidensis MR-1 (formerly S. putrefaciens MR-1) indicated that MR-1, and possibly the other psychrotrophic bacteria isolated appear to not be dissimilatory denitrifiers. In addition, whole genome sequence comparison indicated that MR-1 is more closely related to Vibrio cholerae O1 than to Escherichia coli K12. Finally, a partial microarray containing about 200 genes involved in energy metabolism and regulation were constructed and used to monitor gene expression patterns under anaerobic conditions. Substantial differences in gene expression patterns were observed under aerobic and anaerobic conditions. One interesting observation is that the genes (mtrA, B, and C) involved in metal reduction were highly expressed under both aerobic conditions and anaerobic metal reducing and denitrifying conditions, suggesting that the expression of these genes is not specific to metal reduction. The iron reduction rates in the deletion mutants of mtrB generated by newly developed suicide vectors were much slower than in the wild type strain. The partial microarrays were also used to assess the genome diversity among different metal-reducing bacteria. The results indicated that S. onedensis DLM7 is more closely related to MR-1 than Shewanella sp. W3-6-1. Housing keeping genes and the genes involved in metal reduction appear to be highly conserved between MR-1 and W3-6-1 although the overall genomic diversity is low.

124. Pangenomic Microbial Comparisons by Subtractive Hybridization

Peter Agron, Lyndsay Radnedge, Evan Skowronski, Madison Macht, Jessica Wollard, Sylvia Chin, Aubree Hubbell, Marilyn Seymour, Christina Nocerino, and Gary Andersen

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94550

andersen2@llnl.gov

Sequencing of whole genomes is reshaping microbiology. However, as more sequence information is generated, there will be increased sequence redundancy between closely related species or strains. In the course of time, the amount of new sequence information obtained by whole genome sequencing with current technology will become increasingly less cost efficient. We are exploring the use of suppression subtractive hybridization (SSH) of total DNA as a means of focusing sequencing efforts on unique regions when a reference strain of known sequence is compared to a different isolate of the same species or genus. To rigorously examine this approach, two sequenced strains of Helicobacter pylori (J99 and 26695) were used as a model system, as this allows rapid determination and mapping of difference products based on sequencing alone. Using the high-throughput SSH methods, difference products can be rapidly cloned, sequenced, and then mapped by comparing the data to the H. pylori genome database. To increase the likelihood of amplifying difference products from any given region, several restriction enzymes were used in separate SSH experiments. So far we have obtained data from 2,123 clones that reveal 427 (20%) unique sequences. Control subtractions with an Escherichia coli strain containing the transposon Tn5 against its isogenic parent showed a 270-fold enrichment for Tn5 sequences, demonstrating that SSH is highly effective. Current efforts are focused on: 1) mapping of the difference products onto the relevant genome using the cross_match algorithm and Percent Identity Plots, 2) assessing coverage of the difference regions by the subtracted clones, 3) assessing the redundancy of this coverage and 4) determining the reproducibility of SSH. We will present data that address the overall efficacy of the use of subtractive hybridization for pangenomic microbial comparisons.

125. Prochlorococcus: The Smallest and Most Abundant Photosynthetic Microbe in the Oceans

Sallie W. Chisholm, Gabrielle Rocap, and Lisa Moore

Departments of Civil and Environmental Engineering and Biology; Massachusetts Institute of Technology; 15 Vassar St. 48-425; Cambridge, MA 02139

chisholm@mit.edu

http://web.mit.edu/chisholm/www/

Prochlorococcus is a unicellular cyanobacterium that dominates the temperate and tropical oceans. It lacks phycobilisomes that are characteristic of cyanobacteria, and contains chlorophyll b as its major accessory pigment. This enables it to absorb blue light efficiently at the low-light intensities and blue wavelengths characteristic of the deep euphotic zone. It contributes 30-80% of the total photosynthesis in the oligotrophic oceans, and thus plays a significant role in the global carbon cycle and the Earth's climate. Description of the complete genome of this microbe will greatly advance our understanding of the regulation of these globally important processes.

To this end, colleagues at the DOE Joint Genome Institute (J. Lamerdin et al.) have been working on the complete genome sequence of Prochlorococcus marinus (MED4). The work has progressed rapidly, and the sequence is almost complete. Prochlorococcus is an ideal candidate for complete genome sequencing because (1) it is the smallest known phototroph with a relatively small genome (1.8 Mb), (2) it is widespread and abundant and is easily identified and enumerated in situ using flow cytometry, (3) its unique photosynthetic pigment (divinyl chlorophyll) makes its contribution to total photosynthetic biomass in the oceans easily assessed, and (4) we have an extensive culture collection of isolates from different oceans and environments.

Moreover, we have recently demonstrated that at least two ecotypes of Prochlorococcus coexist in the oceans that are distinguished by their photophysiology and molecular phylogeny. One is capable of growth at irradiances where the other is not. Ultimately, a comparison of the complete genomes of these two ecotypes would provide valuable insights into the regulation of this type of microdiversity in marine microbial systems. In addition, the use of microarray technology for the analysis of gene expression patterns will give us unprecedented insights into how these microbes cope with the dilute environment of the oligotrophic oceans.

126. Sequencing Microbial Genomes of Relevance to Global Climate Change

J. E. Lamerdin¹, K. Burkhart-Schultz¹, A. Arellano¹, S. Stilwagen¹, A. Erler¹, A. Kobayashi¹, M. Shah⁴, D. J. Arp², A. B. Hooper³, S. W. Chisholm⁵, G. Rocap⁶, E. Branscomb⁷, and F. Larimer⁴

¹Joint Genome Institute, Lawrence Livermore National Laboratory, Livermore, CA., ²Botany and Plant Pathology Department, Oregon State University, Corvallis, OR, ³Department of Biochemistry, University of Minnesota, St. Paul, MN, ⁴Oak Ridge National Laboratory, Oak Ridge, TN, ⁵Departments of Civil and Environmental Engineering and Biology, Massachusetts Institute of Technology , Cambridge, MA, ⁶Massachusetts Institute of Technology/Woods Hole Oceanographic Institution Joint Program, Cambridge, MA, ⁷Joint Genome Institute, Production Sequencing Facility, Walnut Creek, CA

lamerdin1@llnl.gov

The Joint Genome Institute (JGI) has established a new program to sequence the genomes of microorganisms that may significantly impact global climate. This effort is focused initially on five microorganisms: Nitrosomonas europaea, Rhodopseudomonas palustris, Nostoc punctiforme, and two marine cyanobacteria, Prochlorococcus marinus and Synechococcus. The common theme shared by these microbes is that all are autotrophic, fairly numerous within their respective ecosystems, and contribute materially to carbon cycling or biomass production (with the exception of N. europaea). By systematic analysis of each genome, we hope to identify specialized nutrient uptake systems, pathways that contribute to or regulate nitrogen fixation, carbon cycling and photosynthesis. With this knowledge, it may be possible to maximize the carbon recycling capabilities of these organisms.

We have completed the initial data generation phase for N. europaea and P. marinus, which yielded >95% of the genomic sequence for each microbe. (Progress towards completion can be monitored through our web site: http://bbrp.llnl.gov/jgi/ microbial/). A similar level of coverage is anticipated for R. palustris by mid-March. Finishing is underway on the first two organisms, and we expect closure by Spring of 2000. The level of coverage achieved by the 'shotgun' phase is readily amenable to generating a rough inventory of the types of genes present in each organism. Preliminary analyses have been performed on N. europaea and P. marinus and the results are available on our web site. The resulting gene 'catalogues' provide the scientific user community access to the contents of unfinished sequence data in a consumable format, without the need for protracted data manipulations on their part. At this meeting, we will present some of the surprising findings that are emerging from the analyses of these two genomes.

This work was performed under the auspices of the U.S. DOE by LLNL under contract no. W-7405-ENG-48.

158. Engineering Deinococcus radiodurans for Bioremediation: Impact of Genomic Sequence

K. S. Makarova^1,2; E. V. Koonin³; H. Brim1, L. Aravind², K. W. Minton¹, L.Tatusov², Y. I. Wolf², O. White³; and M. J. Daly¹

¹Uniformed Services University of the Health Sciences, Bethesda, MD 20814-479, ²The National Center for Biotechnology Information, ³The National Institutes of Health, Bethesda, MD 20814. 3The Institute for Genomic Research, Rockville, MD 20850.

mdaly@usuhs.mil

Extremophiles are nearly always defined with singular characteristics that allow existence within a singular extreme environment. The bacterium Deinococcus radiodurans qualifies as a polyextremeophile, showing remarkable resistance to a range of damage caused by ionizing radiation, dessication, ultraviolet radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is most famous for its extreme resistance to ionizing radiation; it not only can grow continuously in the presence of chronic radiation (6,000 rad per hour), but it can survive acute exposures to gamma radiation that exceed 1,500,000 rad without lethality or induced mutation. These characteristics were the impetus for sequencing its genome and the ongoing development of its use for bioremediation of radioactive wastes. We are using the genomic sequence as a guide to metabolic engineering of D.radiodurans for growth on toxic organic compounds in radioactive sites; and enhancing its survival in nutrient poor radioactive environments. In part, this work is exploiting a newly developed system for analyzing gene expression patterns in D. radiodurans.