Environmental Sciences Division
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Functional Genomics
Funded projects in the ESD Genomics Laboratory currently center around four bacteria of environmental importance (see below). The main focus of these projects is on the use of DNA microarray technology to elucidate gene function and regulation.
Shewanella oneidensis MR-1
Among metal-reducing bacteria, the genus Shewanella is one of the few groups of organisms that have been extensively investigated because of its wide distribution in nature, metabolic versatility, and environmental importance. S. oneidensis MR-1, formerly classified as S. putrefaciens MR-1, is a facultatively anaerobic g-proteobacterium capable of utilizing a wide array of electron acceptors, including oxygen, fumarate, nitrate, nitrite, thiosulfate, elemental sulfur, trimethylamine N-oxide (TMAO), dimethyl sulfoxide (DMSO), iron, and manganese. This organism has attracted considerable research interest due to its potential for bioremediation of metal and volatile organic contaminants in the environment. Recently, the 5-Mbp genome of S. oneidensis MR-1 was completely sequenced by The Institute for Genomic Research (TIGR; http://www.tigr.org/) under the support of the U.S. Department of Energy (Microbial Genome Program and NABIR Program), making it feasible to apply microarray technology to the study of energy metabolism in this bacterium.
Under funding from the DOE Microbial Genome Program, we have initiated genome-wide characterization of S. oneidensis MR-1. In particular, the laboratory is interested in identifying the genes and regulatory networks specifically involved in anaerobic respiration, with emphasis on dissimilatory metal reduction, using DNA microarrays and various proteomic tools. We have established a core team of scientists with expertise in the areas of microbial functional genomics, biochemistry, physiology, ecology, and bioinformatics from Oak Ridge National Laboratory, Michigan State University, California Institute of Technology, NASA Jet Propulsion Laboratory, and Argonne National Laboratory. Initial studies on the genes and regulatory mechanisms underlying respiration in S. oneidensis have been completed using DNA microarrays containing a subset of genes (i.e., 691) with predicted functions in energy metabolism, transcriptional regulation, adaptive responses to environmental stress, iron acquisition, substrate transport, and other cellular processes (see Partial Genome Array). For example, differential mRNA and protein expression profiles of wild-type S. oneidensis MR-1 were investigated under aerobic, fumarate-, Fe(III)-, and nitrate-reducing growth conditions using partial genome arrays, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry (Publications). In addition, insertional mutants of S. oneidensis defective in the Fnr-like etrA (electron transport regulator A) and fur (ferric uptake regulator) genes were generated by suicide plasmid integration and characterized using both genomic and proteomic tools (Publications). We have constructed whole-genome microarrays for MR-1 and further studies are underway.
The Shewanella Federation (http://shewanella.org/), a multi-investigator and cross-institutional consortium, has recently been organized as part of the DOE Microbial Cell Project to characterize the biology of S. oneidensis MR-1 from a whole-system perspective. The Federation will elucidate the transcriptome and proteome of MR-1 under a variety of growth conditions and then use the experimental information to construct predictive models for cellular networks in S. oneidensis MR-1.
Deinococcus radiodurans R1
The ESD Genomics Laboratory is currently investigating the functional genomics of Deinococcus rediodurans R1, a Gram-positive bacterium that was originally identified as a contaminant of irradiated canned meat. All species in the genus Deinococcus, particularly D. radiodurans, are extremely resistant to a number of agents and conditions that damage DNA, including ionizing and ultraviolet (UV) radiation (up to 15,000 Gy), desiccation, and oxidative stress. D. radiodurans is the most radiation-resistant organism described to date, and this phenotype makes it an ideal candidate for studying DNA damage, DNA repair, and resistance to radiation, desiccation, and oxidative stress.
Under the support of the U.S. Department of Energy, the complete 3.3-Mbp genome of D. radiodurans R1 was sequenced by The Institute for Genomic Research (White et al. 1999. Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286:1571-1577). More than 30% of the genes cannot be assigned functional roles. Furthermore, sequence analysis indicates that essentially all of the DNA repair genes identified in D. radiodurans have functional homologs in other prokaryotic species (White et al., 1999), suggesting that the organism’s resistance to extreme DNA damage is possibly attributable to novel genes, pathways, and mechanisms yet to be described. In our laboratory, with support from ORNL’s LDRD Program, we have constructed high-density whole-genome DNA microarrays for D. radiodurans R1 consisting of 3,009 different open reading frames (a genome coverage of 97%). To define the repertoire of DEIRA genes responding to acute irradiation (15 kGy), transcriptome dynamics were examined in the wild type strain R1 using whole-genome microarrays. At least at one time point during DEIRA recovery, 832 genes (28% of the genome) were induced and 451 genes (15%) were repressed two-fold or greater. Genes induced in the early phase of recovery included those involved in DNA replication, repair, recombination, cell wall metabolism, cellular transport, and many encoding uncharacterized proteins. The microarray data suggest that DEIRA cells efficiently coordinate their recovery by a complex network, within which both DNA repair and metabolic functions play critical roles (supplementary data). Part of this work has been published recently in PNAS (see Publications). We are currently using these arrays to identify potentially novel genes involved in the radiation resistance of D. radiodurans. Work on the functional genomics of D. radiodurans is continuing in collaboration with Michael Daly of the Uniformed Services University of the Health Sciences (Bethesda, Maryland) under support from the DOE Microbial Cell Project.
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Supplementary Data
- Supporting Text
- Table A. Master table for normalized whole-genome microarray data. This table is 100 pages and has been formatted to fit on 11 X 17 paper.
- Supporting Text
Rhodopseudomonas palustris
The ESD Genomics Laboratory currently has DOE funding to investigate the functional genomics of Rhodopseudomonas palustris using high-density whole-genome microarrays. R. palustris is a common soil and water bacterium that can convert sunlight to cellular energy, fix carbon dioxide, and generate hydrogen photochemically as a by-product of nitrogen fixation. Recently, the 5.49-Mbp genome of R. palustris has been sequenced by the Joint Genome Institute (http://www.jgi.doe.gov/) as part of the DOE’s carbon management program. The goal of the R. palustris project is to use DNA microarrays to investigate the genes and molecular regulatory mechanisms that are responsible for carbon dioxide fixation and nitrogen fixation (hydrogen production). We are currently in the process of constructing whole-genome microarrays in collaboration with Dr. Caroline Harwood at The University of Iowa. Recently, the DOE funded a proposal led by Robert Tabita of The Ohio State University as part of the Microbial Cell Project to investigate R. palustris from a whole-system perspective using both genomic and proteomic technologies. Our laboratory will be participating in aspects of this project.
Nitrosomonas europaea
Nitrosomonas europaea is an ammonia-oxidizing bacterium and as such, participates in ecosystem carbon and nitrogen cycling. As an obligate autotroph, N. europaea obtains all of its carbon for growth through the assimilation of CO2. As a chemolithotroph, it derives all of its reducing power and energy required for biosynthesis from the transformation of NH3 to NO2-. Of the ammonia-oxidizing bacteria, N. europaea is the best characterized. The organism is of environmental interest, because it carries out nitrification, which influences the concentration of greenhouse gases in the atmosphere, consumes CO2, and initiates the degradation of various environmental pollutants. With the sequencing of the N. europaea genome by the DOE Joint Genome Institute, it is now possible to investigate the total genomic complement of the organism using microarray technology. The goal of this project is to use whole-genome DNA microarrays to study the expression and regulation of genes involved in assimilating CO2 and oxidizing NH3.
Last Modified: March 4, 2003