Microbial Genomics
at the U.S. Department of Energy

DOE Microbial Genome Program Awards 2001

Since its beginning in 1994, the DOE Microbial Genome Program has sparked a revolution in microbiology. From the publication by Craig Venter's group (then at The Institute for Genomic Research, TIGR) of the complete genome sequence of Haemophilus influenza, completed genome sequences of some 51 microbes have been published; sequencing of at least a dozen more are known to be complete but not yet published and sequencing projects of approximately 140 additional microbes are presently known to be in varying stages of progress. Activity in the private sector has also been intense. Sequencing technologies have progressed to the point where a high-throughput facility such as the DOE Joint Genome Institute can draft the sequence of a 2.5 Mb microbe in one day and draft about 65 Mb of microbial sequence (about 17-20 microbes) in one month.

These sequences are enabling a variety of new discoveries. These include new genes and pathways, as well as the insight that the horizontal transfer of genetic information may have been remarkably frequent in microbial evolution. An additional discovery is that even in the 470 gene sequence of the smallest known free-living microbe, Mycoplasma genitalium, perhaps as many as 100 - 150 of those genes are not required for life. One of the most repeatable and astonishing results is that even with numerous microbial genomes finished or nearly finished (and thus sufficient for gene analyses), about 50% of each genome characteristically is comprised of genes of unknown function. Microbes have been isolated (and their genomes sequenced) from environments characterized by extremely low pH, temperatures above boiling water, pressures greater than 200 atmospheres, highly toxic metal concentrations, high radiation fluxes, high salinity, and just about every other inhospitable condition imaginable. It is worth noting that most microbes do not cause diseases and, in fact, their important roles in maintaining the ecology of Earth are becoming clearer.

A completed microbial (or any other) genome represents the source code for life. More tangibly, it represents a list of the parts, working and structural, that a cell (or a multicellular organism) requires to exist and to function. For this reason, closely linked to the Microbial Genome Program, OBER has just launched the Microbial Cell Project. With the assistance of DOE's Offices of Basic Energy Sciences (BES) and Advanced Scientific Computing Research (ASCR), awards have been made initiating research into how microbial cells work and how high-throughput computational technologies can be exploited to model their functioning (see next story). OBER will support research to provide the fundamental understanding that will allow the use of these microbes to address the challenges of carbon sequestration, bioremediation, cellulose degradation, energy production, and biotechnology.

These new Microbial Genome awards represent a departure from the past. Instead of focusing on the high throughput production of new microbial genome sequence data, a mission now given over to the DOE Joint Genome Institute, the foci of the MGP has evolved towards post-genomic analyses. The five thrusts are: A) functional analyses of microbial genomes; B) bioinformatic tools for microbial genome analyses; C) studies of lateral gene transfers, including their frequency and biological constraints; D) novel technologies for genomic characterization; and E) studies of microbial consortia and communities. Of more than 70 applications to the program, 28 awards have been made.

In the area of functional studies, the new awards include:

• John Battista (Louisiana State Univ.) Identifying the Proteins that Mediate the Ionizing Radiation Resistance of Deinococcus radiodurans R1. The objectives of this work are to enhance the understanding of Deinococcus radiodurans and to define the novel family of proteins responsible for DNA damage repair. This will be addressed by identifying genes required for radiation resistance and identifying genes whose expression changes after exposure to radiation. The genes induced by radiation exposure in other radiation-resistant bacteria will be determined as well as any similarities to those seen in D. radiodurans. Even after its genome has been fully sequenced, very little is known about how D. radiodurans tolerates exposure to high does of ionizing radiation.

• Ray Gesteland (University of Utah) Making Sense Out of "Odd ORFs" . Both computational and experimental work will be done to understand the phenomenon of "genome recoding", the determination of additional expressed proteins from a gene that is known to occur by mechanisms such as programmed ribosomal frame shifting and codon redefinition. This phenomenon can result in an underestimate of the proteomic complement of an organism that is predicted from standard genome sequence analysis. This information will strongly impact other disciplines in biology, medicine, and ecology by contributing to a better understanding of genomic information content and expression flexibility under different circumstances. An early focus of this work will be the radiation resistant microbe Deinococcus radiodurans. This issue is an important one in general for translating microbial genome sequences into experimental platforms and tools for meeting the DOE mission.

• David Wilson (Cornell Univ.). Identification and Characterization of Thermobifida fusca Genes Involved in Plant Cell Wall Degradation. This research will elucidate the mechanism, control, and regulation of genes coding for plant polysaccharide-degrading enzymes in Thermobifida fusca, as well as to use the virtually complete genome sequence to study regulation of expression of genes involved in cellulose digestion. This work is expected to contribute to DOE missions in alternate energy source generation such as the conversion of cheap and abundant biomass into fuels such as ethanol and other products, and is consistent with the National Bioenergy Initiative. Additionally, the digestion of plant cell walls by microbes such as T. fusca is of great significance for cycling of carbon in the biosphere and enzymes such as cellulase and xylanase are the initial, and key players in the beginning of such transformations. A final objective is to discover new cellulase genes. Although the aim of this project is discovery of new knowledge, it is also in part a validation of using genome sequence information to discover gene function.

• Derek Lovley (Univ. Massachusetts, Amherst) Improving Functional Analysis of Genes Relevant to Environmental Restoration via an Analysis of the Genome of Geobacter sulfurreducens. This research will identify genes and proteins involved in the matrix of electron transfer systems in the environmentally significant bacterium Geobacter sulfurreducens. The PI and his team will identify the functions of the numerous uncharacterized genes correlating the nutritional or environmental status in which they are expressed, using total genome microarray analyses. Also, a complementary proteomic analysis will be conducted in collaboration with Dr. Carol Giometti at Argonne National Laboratory. Genetic disruption of the genes deemed to be important under a given condition will be used to confirm the function(s) implied. Finally, the previously unknown protein(s) will be isolated and biochemical function(s) determined.

• Brian Palenik (Scripps Inst. of Oceanography). Transport and its Regulation in Marine Microorganisms: A Genomics-Based Approach. This work will explore transport proteins and their regulation in Synechococcus WH8102 and add to our knowledge of nutrient transport in an environmentally important photosynthetic organism. Synechococcus represents an important primary producer, and regulation of transport could have profound environmental importance. The expression of the significant genes that are increased in expression as the organism is put through a series of environmental manipulations, including differing phosphorous, nitrogen, and light regimes, will be probed with microarrays. Then, "knockout mutants" of the transporters will be examined. Comparisons will be carried out with other ocean photosynthetic organisms (whose genome sequences are available) since the activities of these microbes can significantly affect the biosphere.

• Caroline Harwood (Univ. of Iowa). Use of DNA Microarrays for Understanding the Genetic and Metabolic Regulation of Carbon Dioxide Fixation and Hydrogen Production in Rhodopseudomonas palustris. This research will use new microarray technologies to determine genes expressed during carbon dioxide and nitrogen fixation (closely linked to hydrogen production) by the photosynthetic bacterium, Rhodopseudomonas palustris. The complete genome of R. palustris was recently completed at DOE's Joint Genome Institute; the full genome contains 4,690 predicted protein-encoding genes, and all of these will be studied to assess their contribution to carbon dioxide and nitrogen fixation. Since ~40% of the genes deduced from the genome sequence have not been assigned a function, it is likely that some of these will be implicated in the array analysis of carbon and nitrogen cycle functions. This project will provide very useful information on pattern of gene expression, from a whole genome perspective, as they relate to carbon and nitrogen flux in a metabolically versatile model organism. Such information will be useful in the future for attempts to engineer carbon and nitrogen fixation pathways.

• Dan Arp (Oregon State Univ.). Gene Expression Profiles in Nitrosmonas europaea, an Obligate Chemolithoautotroph. The goal of this research is to describe in Nitrosomonas europaea the gene regulatory networks that respond to changes in nutrients and other conditions, and to infer gene function, using microarrays to investigate growing vs. resting cells under a variety of nutrient conditions. The Arp lab will also determine changes in gene expression due to changes in conditions, nutrients, or pollutants, and to identify regulatory genes and determine their function by mutagenesis. N. europaea, as a nitrifier, plays an important role in both the carbon and nitrogen cycles and thus in agriculture. The genome of N. europaea has been sequenced essentially to closure, and this work will focus on patterns of expression using microarrays constructed with all 2500 genes. This project will be of great significance to those concerned with understanding a tractable model organism that plays a pivotal role in the global nitrogen cycle, contributes to greenhouse gas production, and produces water soluble oxides of nitrogen that can become groundwater.

• Jizhong Zhou (Oak Ridge National Laboratory) Genome-Wide Functional Analysis of the Metal Reducing Bacterium Shewanella oneidensis MR-1. This project will construct whole genome microarrays for the genomes of several key DOE mission-relevant microbes, including Shewanella oneidensis MR-1, Rhodopseudomonas palustris, and for Nitrosomonas europaea, and use these microarrays to perform experiments to elucidate gene expression profiles under different growth conditions as well as to characterize different genetic mutants. The S. oneidensis work will be carried out in collaboration with Drs. Jim Tiedje (Michigan State Univ.) and Ken Nealson (Univ. Southern California). The R. palustris work will be carried out in collaboration with Dr. Caroline Harwood at the University of Iowa and the N. europaea work will be carried out in collaboration with Dr. Dan Arp at the Oregon State University.

In the area of bioinformatics tools, resources, and activities, the new projects include:

• Owen White (TIGR) Continuation of the Comprehensive Microbial Resource. The Comprehensive Microbial Resource (CMR) is a coherent collection of careful, consistent annotations of microbial genome data. This resource develops and maintains standard operational procedures for annotation of microbial genomes, provides an annotation engine for use on microbial sequence data from non-TIGR genome sequencing centers, organizes all genes from microbial genomes into an array of orthologous gene families and presents a Web interface that allows full access to the CMR. The CMR integrates all of TIGR's considerable microbial sequencing and bioinformatics expertise in an easy to use web interface. The consistent annotation of genomic data is the key to its wide use and powerful application and this project will expand the CMR beyond just TIGR-sequenced genomes.

• Mark Borodovsky (Univ. Georgia). New Markov Model Approaches to Deciphering Microbial Genome Function and Evolution. This project will enhance existing gene finding algorithms to improve detection of atypical genes in microbial genomes. These atypical genes will be analyzed to identify those that may be putative laterally transferred genes. The PI will use phylogenetic reconstruction methods to test the possibility that each atypical gene is a result of lateral gene transfer, to determine the derivation of the lineage, and to estimate the overall rate of lateral gene transfer, particularly with respect to phylogenetic and/or ecological separation between donors and recipients. The putative functional roles of these atypical genes will be determined to better understand what types are most prone to be identified as atypical and those that are the most likely to been transferred between species. Understanding microbial plasticity is a high priority since the identification of laterally transferred genes is changing our view of the bacterial genome and add substantially to the evolving concept of a bacterial species. It will also add to our understanding of the evolution of life on this planet.

• Jeremy Edwards (Univ. of Delaware). Metabolic Engineering of Deinococcus radiodurans Based on Computational Analysis and Functional Genomics. This research will develop novel computational and high-throughput experimental tools to analyze the DNA repair pathways in Deinococcus radiodurans and the influence of the metabolic flux distribution on DNA repair. While D. radiodurans can survive extremely high doses of radiation when grown under nutrient-rich conditions, its DNA repair capabilities are impaired under nutrient-poor conditions. The PI and coworkers will construct a metabolic flux balance analysis model based on the genome of D. radiodurans, and establish a dynamic model of the known DNA repair pathways.

• Charles Lawrence (Wadsworth Center). Identification and Characterization of Transcription Networks Environmentally Significant Species. This work will develop technologies for the identification of transcription regulation networks in genomes of microbes sequenced by DOE-funded genomic projects. The PI will develop specialized algorithms to identify all transcription factors in several genomes and group them into families based on homology. The goal ultimately is a complete characterization of the network of transcriptional regulation for several environmentally important organisms. The approach will include cross-species comparisons, clustering of transcription factor binding sites and experimental validation using a robotic 96 column assay system.

• Gary Olsen (University of Illinois). Bioinformatics for Genome Analysis. This research will integrate the tools and concepts of molecular phylogenetics with those of molecular biology as the basis for building an integrated computational tool to construct and edit multiple sequence alignments. This will enhance the ability to add data from new genomes without recomputing the entire alignment and evolutionary tree, while maintaining manual edits with priority over gaps and residue placements with automatic rules. Development of tools for genomic analysis, such as described here will benefit the entire community of researchers involved in analysis of genomic data.

• Monica Riley (Marine Biological Lab). Development of Metabolic Pathway Database for the Metal Reducing Bacterium Shewanella oneidensis MR-1. This project will construct a metabolic database for Shewanella oneidensis, examine proposed pathways for missing functionality, experimentally validate the pathways, and attempt to identify candidates for the missing functions. This will be done by: a) comparing Shewanella with the EcoCyc database (a well-known database of metabolic pathways and other information for the model organism E. coli); b) identifying pathways unique to S. oneidensis; c) using microarrays to identify "gap" proteins; and d) using mutagenesis to identify putative genes. The results of this research will contribute useful genomic data not only to those interested in S. oneidensis, but also to a wider research community, including micro-, molecular-, and cellular-biologists.

• Larry Wackett (University of Minnesota). Functional Analysis and Discovery of Microbial Genes Transforming Metallic and Organic Pollutants. This work will be an extension of the University of Minnesota Biocatalysis/Biodegradation Database (UM-BDD) to incorporate data on microbial metabolism relating to metals, metalloids and organometallics. The UM-BBD will also include a significant effort to extract and sequence enzymes with heretofore unknown functions relating to metals, metalloids and organometallics leading to the identification of microbial genes involved in the transformation of metallic and organic pollutants. The UM-BBD is distinct from other metabolism databases in that it focuses specifically on catabolism of toxic compounds and thus will support the accumulation and organization of knowledge on biodegradation. The database portion will be closely modeled on the present (and widely used) UM-BBD database.

• Diane Makowski (Argonne National Lab). Novel Combinatorial Biology Method to Functionally Characterize Microbial ORFs. This project aim is the genome-wide identification of small molecule binding sites on microbial proteins. The PI will use phage-display technology to express and to identify small peptides that bind ligands. These peptides will be used to determine both consensus ligand-binding motifs as well as the proteins containing these motifs. This information will then be used to annotate the functionally responsible genes. Computational analyses will then be applied to annotate genes from other sequenced microbial (and other) genomes, in particular to reduce the large fraction of genes that remain unannotated by current approaches.

• George Garrity (Michigan State University). Application of Exploratory Data Analysis Techniques for Visualization of Extremely Large Sequence Data Sets. This research will be carried out by the editors of the Bergey's Manual, using a variety of statistical analyses to view information on bacterial species so that the species can be clustered functionally and evolutionarily. The core idea is that phylogenetic trees based on different molecules can give different phylogenies revealing patterns in the evolution of microbial genes and genomes. The results of this work will include a dynamic atlas of taxonomic/phlyogenetic maps (i.e. base maps that can be overlaid with phenotypic and other data), and an up-to-date checklist of prokaryotic species (e.g. sequence accessions and taxonomic placement). Robust knowledge of phylogenetic and taxonomic relationships is clearly critical for comparative genomics studies that are basic to functional characterization of genes and proteins.

• William Cannon (Pacific Northwest National Lab) Bioinformatics Tools to Define the Proteomic State of the Microbial Cell. The major aim of this research will be the development of computational tools for the analysis of the output from high throughput Mass Spectroscopy of microbial proteins, in association with Dick Smith at PNNL. This is key to the discovery of post-translational protein modifications, unidentified open reading frames, mutations, sequencing errors, and protein expression levels. An early microbial target will be the fully sequenced and highly radiation-resistant microbe Deinococcus radiodurans.

In the area of studies of horizontal gene transfers, the new projects include:

• Gary Olsen (Univ. Illinois). Gene Transfer: Past and Present. The PI and his colleague, Dr. Carl Woese will explore horizontal gene transfer to: 1) estimate the frequencies of recent gene transfers to diverse microbial lineages; 2) determine whether there are genomic, evolutionary or lifestyle traits that correlate with high acquisition rates of external genetic information; 3) identify donor lineages of ancient gene transfers; and 4) determine any relationship between gene transfer and biologically important events such as separation of the major lineages, to better define the shape and character of the evolutionary tree. In contemplation of theories of evolution, genome remodeling by horizontal gene has to be a major factor. On a practical scale, horizontal transfer may underlie the rapid transfer of antibiotic resistances and is a constant concern for the containment of genetically engineered microorganisms. It is clear that this work will contribute to the general understanding of lateral transfer in evolution and to the genome stability of microbes. Careful study of lateral gene transfer (LGT) among these organisms will certainly lead to insights about the nature of LGT and how these organisms can exchange genetic material to increase survival during environmental stress.

• Karen Nelson (TIGR). Gene Transfer and Hybridization Studies in Hyperthermophilic Species. Recent findings from whole genome sequencing of Thermotoga maritima and high temperature archaea show extensive genomic homologies that could have only arose by lateral gene transfer (LGT). The objective of this work is to apply biochemical methods to identify regions that are common and those that are unique among the genomes of approximately 50 high temperature bacteria and archaea. While similarity at the DNA sequence level is the ultimate indicator of relatedness among DNA sequences, this work will facilitate examination of a large number of genomes avoiding whole genome sequencing efforts. In addition, the PI's will examine the linkage between Insertion Sequence (IS) elements and putative laterally transferred regions as an indication for the involvement of transposition in transfer events. They will also attempt to transfer genes between Thermatoga strains by conjugation, transformation and recombination. While anecdotal evidence for lateral gene transfers exists, the work proposed here will represent a thorough examination of this phenomenon. The results could have a profound influence on our understanding of the role of LGT in the evolution of microbes representing early-evolving lineages. In particular, the extent of, and possible natural constraints on, the occurrence of LGT will be explored by this research.

• Terry Marsh (Michigan State Univ). Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation. This work will investigate genomic plasticity in two environmental isolates: Ralstonia eutropha and R. pickettii. Both organisms are representative of a group of widely-distributed bacteria that are important in processes such as hydrocarbon degradation and resistance to high concentrations of metals. In addition, there is evidence for a mutator phenotype among some isolates in these two species. The overall goal of the project is to understand the dynamics of the genome in the natural environment. To this end, the project will: (1) identify patterns of genomic change among evolved lineages of R. eutropha TFD41, (2) identify sequences surrounding junctions where deletions/rearrangements have occurred in order to identify controlling elements, (3) identify differences in genomic structure between two sub-populations of R. pickettii, and (4) determine if genomic rearrangement elicited by environmental stress accelerates the rate of evolution. The question of how genetically stable the bioremediation strains are and what, if anything, is the risk of genomic plasticity, will definitely arise and needs to be addressed as soon as possible.

• Howard Ochman (University of Arizona). Lateral Gene Transfer and the History of Bacterial Genomes. This project will investigate a set of 30 conserved and universally distributed genes to assess their involvement in lateral gene transfers (LGT) and to establish the effects of lateral gene transfer on shaping bacterial genomes. This work will assess how well the standard 16s rRNA-based phylogeny represents the relationships among bacteria as revealed by common genes and whether there is a core set of genes that is "immune" from LGT. Additionally, this project will explore which genes are transferred and what phylogenetic limits to LGT may exist, if the transferred genes are functionally related or genetically linked, and what proportion of bacterial genomes arose by LGT and what factors influence that proportion.

In the area of novel technologies, the new projects include:

• David Schwartz (Univ. Wisconsin Madison). Microbial Sequence-Ready Maps by Optical Mapping. The goal of this effort is to characterize by optical mapping ~12 microbial genomes over a three year period. This effort will be closely coordinated with the selection of microbes targeted for the high-throughput DNA sequencing effort at the Joint Genome Institute (JGI). These microbes represent a wide range including those involved in photosynthesis, anaerobic respiration, and iron oxidation. The Schwartz lab will construct optical maps for the DOE microbe portfolio and link these maps with existing sequence information and physical map resources. The push of optical mapping into a higher-throughput production operation for sequence assembly scaffolding, assisting assembly, and verifying finished sequences is greatly needed into the genome community. This effort will greatly benefit DOE's efforts to sequence the genomes of additional organisms whose genomes are not readily sizeable by traditional methods. One of the first planned targets is the genome of Thalassiosira pseudonana, a diatom.

• Jay Keasling (Univ. California Berkeley). Physiomics Array: A Platform for Genome Research and Cultivation of Difficult-to-Cultivate Microorganisms. In this work, arrays manufactured using chip micro-fabrication technology will be developed that will enable the automated testing of a large range of culture conditions. In this way, difficult-to-grow organisms can be exposed to different environmental conditions to discover their growth requirements and the effects of different metabolic substances on the physiology of established cultures can be extensively tested. The bottom of each culture cell has integrated sensors, heating elements and an oxygen generation unit. Once developed, the devices can be produced cheaply on a large scale. The development of a refined, high-throughput cultivation device (and support systems) will permit exploration of cultivation parameters much more efficiently than currently possible. Thus, it will serve as a tool for improving our understanding of microbial physiology of both known and yet undiscovered microbes.

In the area of studies of microbial consortia and hard-to-culture species, the new projects include:

• Jill Banfield (Univ. California, Berkeley). Genomic Analysis of a Spatially and Geochemically Confined, Low Diversity Microbial Community. The goal of this work is to characterize both the genetics and biochemistry of a community of acid-tolerant microbes and to study lateral gene transfer events between members of this community using sequence information gained in association with the DOE Joint Genome Institute. The characterization of metabolism and the biochemistry of members of this extraordinary community of microbes will utilize the BAC libraries made by Dr. Ed DeLong and will give insights into the nature of some of the microbial community interactions that permit this consortium to thrive.

• Fred Brockman (Pacific Northwest National Lab). Assembly and Analysis of Microbial Subgenomes From Undefined Consortia. This work will explore microbial sub-genomes from undefined microbial consortia in contaminated subsurface sediments and characterize a genomic "signature" for the microbes in these environments in highly radioactive aquifer sediments. Using an approach similar to the Banfield work cited above, this study aims to explore the utility of assembling large genomic stretches from uncultivable microorganisms using overlapping BAC libraries, and 2) annotate and analyze the sub-genomes to provide insight into their genomic content and functional capabilities. Preliminary analyses of the contaminated Hanford sediments suggest that the microbial flora contains members that are poorly (or not at all) represented in culture collections. This work should provide important information on metabolic capabilities of the microbial community residing in highly radioactive sediments, a priority DOE research area.

• Cheryl Kuske (Los Alamos National Lab). Identification and Isolation of Active, Noncultured Bacteria From Radionuclide- and Metal-Contaminated Environments for Genome Analysis. Cultured bacteria are only a small proportion of the microbial world. Noncultured species comprise a huge reservoir of potential new genes that could address DOE mission needs. This research will continue the PI's work to determine the abundant and active microbial species in radionuclide and metal contaminated soils and sediments and to flow sort such cells to enable assessments of their genetic diversity. Additional outcomes will be the discovery of new genes, and the development of methods for amplification of DNAs from such microbes to generate libraries for future sequencing analyses. This will allow further characterization of microbial backgrounds as well as the isolation of useful genes.

• David Kirchman (Univ. Delaware). Metagenomic Analysis of Uncultured Cytophaga and Beta 1,4 Glycanases in Marine Consortia. This work will address a key aspect of carbon cycling in the biosphere--how insoluble biopolymers (cellulose, xylan, chitin, for example) are broken down to enter the food chain. This work will focus on microbes in the "Cytophaga-Bacteroides" groups and their key roles in biopolymer degradation. The project will look at specific coding sequences in the genomes of members of the hard-to-culture marine bacteria, Cytophagales. The Cytophaga are now known to be widespread in the environment, but not much is known about their activities. They may well be dominant organisms in the degradation of polymers. This work will explore ways to use sequence data to probe microbial physiology, a necessary direction to take in the post-genomics era. This work will push the limits of deducing physiology from sequence data. Ultimately, data from studies such as this may be used to improve models of carbon cycles in the world's oceans.

The impact of the DOE Microbial Genome Program has been great. Genome sequencing projects are multiplying, not least because, with the approaching conclusion of the Human Genome Project, the US has considerable sequencing capacity that provides an immense opportunity for biology. In fact, our capacity to sequence outstrips our capacity to understand all the information in the sequences we have acquired. This is no reason to stop however; as the sequence database grows, so does its utility for comparative genomic purposes, for gene discovery, for understanding the biology of our environment, and perhaps most powerfully, for fueling the engine of biological science by generating testable hypotheses that will keep biologists busy for years to come.

Daniel Drell with contributions from John Houghton and Anna Palmisano (October, 2001).