1999 RESEARCH PROJECTS Program Element 3 Biomolecular Sciences and Engineering PROJECT: Optimizing a Metalloregulator for Metallosequestration and Metallosensing PRINCIPAL INVESTIGATOR: Anne O. Summers PROGRAM ELEMENT 3 Biomolecular Science and Engineering The objectives of this project are: Define the biochemical and biophysical basis for the metal affinity and specificity of merR; re-design MerR to novel specificity and high affinity for radionuclides and processing waste metals; and test an optimized version of the MerR MBD domain for its ability to protect cells expressing it. The merR loci will be developed so that producing strains would display increased resistance owing to aggressive sequestration of metal by merR protein. This would give bacteria an increased range of operation in metal contaminated sites. The engineered merR loci will also be transferred into Deinococcus radiodurans. This would increase the useful range to radionuclide contaminated areas. Genetic manipulation of the merR should provide both basic and applied insights . PROJECT: In-Situ Survival Mechanisms of Sulfate-Reducing Bacteria in Polluted Sediments PRINCIPAL INVESTIGATOR: Lee R. Krumholz PROGRAM ELEMENT 3 Biomolecular Science and Engineering This work seeks to identify genes of Desulfovibrio desulfuricans that respond to environmental stress. Its specific goals are to: Develop a genetic system to modify and tag specific genes in the sulfate reducing bacterium Desulfovibrio desulfuricans. Using tagged genes, determine which genes are important for activities in either normal or contaminated subsurface sediment. Establish function of these genes 4. Link genotype to phenotype. The primary technique is Signature Tagged Mutagenesis (STM). The STM method is a relatively new approach to defining genetic loci that are critical to survival or fitness of an organism under a controlled set of environmental conditions. This application will provide valuable information on the survival of sulfate reducing bacteria under field conditions. PROJECT: Flow Cytometry Technique for Multiplexed Detection, Quantification, and Isolation of Nucleic Acids PRINCIPAL INVESTIGATOR: Mary Lowe PROGRAM ELEMENT 3 Biomolecular Science and Engineering The goal of this work is to develop a new polystyrene bead-based capture method to identify specific DNA sequences from mixtures of heterogeneous DNA samples obtained from environmental samples. The methodology uses beads impregnated with different color fluorescent dyes. The beads are usually coupled to DNA oligonucleotides that are used as capture probes. Following capture of complementary DNA sequences from the environmental samples, the fluorescent beads can be analyzed and separated by flow cytometry. The bead-based method may provide information comparable to information obtained using DNA microarrays, though with potential advantages such as greater sensitivity, quantitation, and sequence determination. The method can also be preparative; primers based on the capture probe sequences and universal anchor primers can potentially be used to PCR amplify the captured DNA fragment for cloning and sequence analysis. In this application, the investigators propose to develop the technology for the identification of DNA fragments from microorganisms that may be involved in metal bioremediation. The specific aims for one year are as follows. Evaluate the bead-based method with respect to sensitivity, sequence discrimination, accuracy and precision in measuring abundances. Determine if the bead-based method can measure abundances of seven genera of bacteria, previously identified to be important for metal reduction. Develop an assay using beads to capture useful genes from environmental samples and prepare the captured material for PCR amplification, cloning, and sequencing. Assemble a set of candidate probes for metal reduction genes, and determine if the bead-based method can expedite the development of effective probes for capturing these genes. The sequence length of the capture probe may need to be considered. PROJECT: Mechanisms for Uranium and Technetium Reduction inGeobacter Sulfurreducenss PRINCIPAL INVESTIGATOR: Jon Lloyd PROGRAM ELEMENT 3 Biomolecular Science and Engineering This work will investigate and identify biochemical and genetic components of U(VI) and Tc(VII) reduction by G. sulfurreducens. Inasmuch as the microbial components of these reducing pathways have not been elucidated, this work would contribute to progress in the field. The objectives are: to localize and purify components involved in U(VI) and Tc(VII) reduction in Geobacter sp., to identify the genes for the reductase components, to verify physiological relevance of these reductases, to determine if identified mechanism of reduction is shared by other microorganisms. These objectives build on previous work on this organism (Geobacter sulfurreducens) including genome sequencing that will soon be finished. These experiments will lead to an understanding of the mechanisms involved in U(VI) and Tc(VII) reduction and that this in turn will provide insight into the development of strategies to promote in situ reduction of these metals. PROJECT: Molecular and Microcosm Analyses of the Potential for Gene Transfer in Radionuclei and Metal-Contaminated Subsurface Environments PRINCIPAL INVESTIGATOR: Tamar Barkay PROGRAM ELEMENT 3 Biomolecular Science and Engineering This project will address whether metal resistance in subsurface soils evolved by conjugal transfer, compare gene transfer in pristine and contaminated subsurface microbial communities, determine if gene transfer is affected by the presence of metals, and determine whether the spread of metal resistance genes increases the communities' resistance and diversity during metal stress. This will be done by assessing the role of horizontal gene transfer as it relates to metal resistance and metal transformation in microbial populations. The PI will combine a retrospective approach, defined as the detection of horizontal gene transfer leading to the evolution of a specific phenotype, and a prospective approach, defined as the measurement of gene transfer using experimental systems, to establish the role of horizontal gene transfer in the evolution of metal resistant microbial populations. The specific aims of the application are to: determine if metal resistances among subsurface microbes has evolved by gene transfer, assess the potential for gene transfer in subsurface microbial communities; measure the rates of gene transfer in a subsurface soil microcosm; and, determine if gene transfer increases the ability of a microbial community to respond to metal stress. PROJECT: Probing The Proteome With Capillary Isoelectric Focusing-Esi Fticr Mass Spectrometry PRINCIPAL INVESTIGATOR: Richard D. Smith PROGRAM ELEMENT 3 Biomolecular Science and Engineering Bacterial strains such as Shewanella putrefaciens strain MR-1 are key organisms in the bioremediation of metals due to their ability to enzymatically reduce and precipitate a diverse range of heavy metals and radionuclides. Important in these processes is the need to develop improved enzymatic pathways in these organisms. As a first step, the proteome of the organism must be completely characterized. The proteome is defined as the entire protein complement of the cell expressed under a given set of conditions. A single genome can exhibit many different proteomes depending on stage in cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress, etc.), or the manifestation of disease states. While the availability of full genomic reference sequences provides a set of road maps as to what is possible, and measurements of the expressed RNAs tells us what might happen, the proteome is the key that tells us what really happens. Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes, which requires faster and more sensitive capabilities for the characterization of cellular constituents. We are currently developing technologies that will allow the visualization of the protein complement by obtaining comparative displays for the expression of many proteins simultaneously, based upon stable-isotope labeling. Two versions of each protein are generated and analyzed simultaneously, to precisely establish changes in expression. Capillary isoelectric focusing on-line with Fourier transform ion cyclotron resonance mass spectrometry provide a powerful tool to study the changes in expression (i.e., repression or induction) for hundreds of proteins simultaneously. Further characterization of the proteome can be accomplished by characterization of the proteolytic fragments of the proteins in the organism. For many proteins, these proteolytic fragments can be used as unique mass markers for the identification of the proteins in question. Additionally, the sequence of the peptides can be determined as another identification technique. These combined technologies will enable ultra-sensitive proteome ­wide expression profiling to evaluate changes in the complete proteome of the iron reducing bacterium Shewanella putreficiens strain MR-1 induced by switching from aerobic to anaerobic respiration with heavy metals and radionuclides. PROJECT: Characterization of Environmental Regulation of the Genes and Proteins Involved in Metal Reduction Pathways in Shewanella Putrefaciens PRINCIPAL INVESTIGATOR: Carol S. Giometti PROGRAM ELEMENT 3 Biomolecular Science and Engineering Shewanella putrefaciens is a versatile microbe capable of metal reduction in both aerobic and anaerobic environments and is thus of interest for bioremediation of waste sites containing toxic metal compounds. Assessing the usefulness of S. putrefaciens for bioremediation, however, requires characterization of the molecular mechanisms and regulation of the metal reduction activity. The U.S. Department of Energy Microbial Genome Program is funding the sequencing of the S. putrefaciens genome and the development of microarrays containing all ORFs from S. putrefaciens MR-1. NABIR is now funding a project to correlate the output of these Microbial Genome projects with changes in protein expression as a means to characterize the regulatory mechanisms controlling metal reduction activity when S. putrefaciens is grown in different environmental conditions. Shifts in the abundance of specific proteins are indicative of gene regulation, while the relative abundance of chemically modified forms of proteins (i.e., phosphorylated, glycosylated, deamidated, or methylated) reveals mechanisms of metabolic pathway regulation. In this new NABIR project, S. putrefaciens MR-1 cells are grown under experimental conditions designed to replicate metal contamination in a variety of pH and temperature environments. Messenger RNA and proteins are extracted from the cells and analyzed using microarrays and two-dimensional gel electrophoresis, respectively. Proteins altered in abundance in cells grown in the presence of metals will be identified and the correspondence between protein changes and changes in the expression of specific genes detected through the microarray analysis will be examined. Post-translational modifications will be characterized and the mechanism of regulation of protein function deduced. Based on identified changes in gene expression in different environmental conditions, knockout mutants will be generated to determine whether the regulated genes are essential for cell survival. The results of the proposed experiments will identify (1) gene sequences required for S. putrefaciens' metal reduction activity in a variety of environments, (2) regulatory pathways controlling the abundance of the gene products, and (3) the effects of those genes on cell viability. PROJECT: Cellular Response of Shewanella Putrefaciens to Soluble and Solid-Phase Metal Electron Acceptors PRINCIPAL INVESTIGATOR: Margaret Romine PROGRAM ELEMENT 3 Biomolecular Science and Engineering The fate and transport of many multivalent metals and radionuclides can be strongly influenced by a phylogenetically diverse group of microorganisms, termed dissimilatory metal reducing bacteria (DMRB). The DMRB, Shewanella putrefaciens MR-1, a facultative anaerobe that displays remarkable respiratory capacity, is amenable to genetic manipulation and is the subject of a DOE sponsored microbial genome sequencing project. The research proposed herein is intended to develop an understanding, at the genetic level, of how S. putrefaciens derives energy by coupling oxidation of organic compounds or H2 to reduction of either soluble- and solid-phase Fe(III) oxides. In particular, we intend to investigate specialized functions that we hypothesize are required for utilizing solid phase Fe(III) (oxides or oxyhydroxides). Hypotheses will be tested through the study of differential transcriptional responses associated with the growth and/or respiration of S. putrefaciens MR-1 in the presence of soluble- or solid-phase (such as Fe oxides) electron acceptors. Transcriptional activity will be measured by the use of cloned Shewanella promoters fused to the GFP reporter gene. Time-course measurements will reveal the sequence of transcriptional events that mediate the response of S. putrefaciens to anaerobic respiration via dissimilatory iron respiration. This research will provide important insights into the response of S. putrefaciens, at the cellular level, to metals, both soluble- and solid-phase, as electron acceptors. Ultimately, we expect to gain insights into the mechanisms by which this metabolically versatile organism accesses Fe(III) for respiration from insoluble metal oxides. [Back to Award Recipients Page]