2003 RESEARCH PROJECTS Biomolecular Sciences and Engineering PROJECT: Analysis of Shewanella oneidensis Membrane Protein Expression in Response to Electron Acceptor Availability PRINCIPAL INVESTIGATOR: Carol S. Giomettii Biomolecular Science and Engineering Shewanella oneidensis MR-1 is a facultative anaerobe capable of reducing a variety of compounds under anaerobic conditions, including nitrate, manganese, iron, fumarate, chromate, and uranium. The reduction of chromate and uranium is particularly relevant to the development of bioremediation strategies for immobilization of toxic metal compounds, a primary goal of the U.S. Department of Energy NABIR program. Funded by the NABIR program, and in parallel with the Microbial Genome Program’s MR-1 genome sequencing and microarray development, we have monitored patterns of protein expression in MR-1 total cell lysates grown with oxygen, nitrate, iron, and fumarate. Regulation of specific S. oneidensis genes and proteins related to energy metabolism, cell division, and membrane transport of specific molecules has been observed in response to electron acceptor availability. With these data as a foundation, we now propose to focus on the proteins associated with the MR-1 membrane, the cell compartment directly in contact with the metals of interest. We hypothesize that the flexibility S. oneidensis MR-1 exhibits in utilization of diverse terminal electron acceptors lies in the microbe’s repertoire of potential electron transport pathways and the regulation of their expression. We propose that comprehensive identification of all of the cytoplasmic and outer membrane proteins expressed by wild type MR-1 and MR-1 mutants deficient in specific outer membrane proteins, through the use of surface labeling, subcellular fractionation, and both electrophoresis and mass spectrometry protein separation methods, will elucidate the specific components of the electron transport pathways responsible for reduction of different metals, including chromate and uranium. PROJECT: Parallel Proteomic Identification of Metal Reductases and Determination of their Relative Abundance in a Series of Metal Reducing Microbes PRINCIPAL INVESTIGATOR: Mary Lipton Biomolecular Science and Engineering Central to the NABIR goal to develop the scientific basis for in situ radioactive groundwater contaminant remediation is the fundamental understanding of microorganisms with dissmilatory metal reducing activity. In order to effectively exploit these bacteria, it is necessary to know which enzymes and pathways are involved. Additionally, it would be advantageous to understand the similarities and differences of these pathways across different bacteria for effective deployment in bioremediation, as well as to identify new microbes capable of such activities. Most approaches to identify these enzymes or enzyme complexes rely on biochemical purification to homogeneity with subsequent N-terminal sequencing of digested peptides. However, loss of activity before achieving purity often necessitates repetition of the entire process. Newly developed proteomics capabilities at PNNL allow for the identification of many proteins from a single sample through mass spectrometry analysis. Thus the need for absolute sample purity is eliminated, and potential enzymatic targets for metal reduction are reduced to a small subset of proteins whose metal reduction activity can be related by genetic manipulation. In the proposed research, we will use high throughput proteomics to identify the proteins responsible for metal reduction activity across 5 organisms: Shewanella oneidensis MR-1, Geobacter sulfurreducens, Pseudomonas fluorescens, Desulfovibrio desulfuricans G20, and Deinococcus radiodurans allowing inferences to be made as to the similarities and differences of activities throughout the organisms. PROJECT: Comparative biochemistry and physiology of iron-respiring bacteria from acidic and neutral pH environments PRINCIPAL INVESTIGATOR: Tim Magnuson   Biomolecular Science and Engineering Acidophilic dissimilatory iron-reducing bacteria (DIRB) are now being detected in a variety of low-pH habitats where Fe(III) reduction is taking place, and may represent a significant proportion of metal-transforming organisms in these environments. Although a great deal of effort has been put into understanding the biochemistry and physiology of neutrophilic DIRB, almost nothing is known about their acidophilic counterparts. A systematic study of the biochemical mechanisms of iron reduction in acidophilic DIRB is now in order, and there are several representative organisms available for study. In addition, the NABIR Field Research Center is available for obtaining samples for enrichment and isolation of additional representatives of acidophilic DIRB. Studies in this area would reveal novel biochemical mechanisms that acidophilic DIRB utilize to respire on iron. Direct comparison of acidophilic and neutrophilic organisms will be feasible, and may reveal fundamental differences in attachment mechanisms and enzyme-mediated electron transport. PROJECT: Starvation promoter-driven metal and radionuclide Bioremediation in combinatorial bacteria PRINCIPAL INVESTIGATOR: A.C. Matin Biomolecular Science and Engineering Our long-term goal is to improve bioremediation at the DOE waste-sites. These contain a complex mixture of contaminants, which inhibit the enzymes important in bioremediation as well as the bacterium (Pseudomonas putida) harboring them. Our approach is protein and genetic engineering to generate more efficient enzymes and bacterial strains for bioremediation. To date, we have focused on chromate [Cr(VI)] bioremediation. This heavy metal is toxic and mobile and thus its presence at the waste sites threatens to contaminate drinking water supplies. Bacteria can reduce it to a nontoxic form [Cr(III)], which is also insoluble and can therefore be effectively sequestered. Bacteria possess multiple mechanisms for reducing chromate. Some are non-enzymatic, such as the reduction mediated by glutathione. This reduction, as well as those carried out by several cellular enzymes, involve one-electron reduction of Cr(VI). The result is generation of Cr(V), which has a high propensity to react with molecular oxygen, resulting in the production of reactive oxygen species (ROS). In such reactions, chromate acts essentially as a shuttle for the generation of large amounts of ROS. The latter can damage vital cell macromolecules incapacitating or killing the remediating bacterium. We thus attempted to find bacterial enzymes that would convert Cr(VI) to Cr(III) with minimal ROS generation. In our search, we looked for three additional characteristics: a) possession of stress-enhancing properties, so that improving their activity will simultaneously also bolster bacterial survival at the stressful DOE sites; b) a broad substrate range, so it might be easier to engineer proteins from these enzymes that would be effective in remediating also other heavy metal and radionuclide contaminants of the sites; and c) regulation by mechanisms that promoted effective expression under the stressful site conditions. We found two gene families (widely distributed in bacteria) and demonstrated, using four cloned genes and proteins of these families, that they possessed the above characteristics. Studies dealing with the mechanistic aspects of these enzymes demonstrated that they generated minimal ROS during Cr(VI) reduction because they reduced this compound by an obligatory (or nearly so) two-electron reduction; that their biological role is to act as an antioxidant defense; and that their high level expression under stressed conditions resulted from their regulation by starvation promoters. We have also identified targets for improvements: a) higher activity and affinity for chromate, while retaining a tight two-electron reducing capability; b) higher expression levels under stressed conditions and improved activity of the enzymes within the cell under these conditions; and c) improved resistance to co-contaminants of the waste sites for both the enzymes as well as P. putida. Gene-, family-, and genome-DNA shuffling are being used to obtain improved proteins and bacteria. Screening procedures have been designed, involving high-throughput microtiter plate methods and the use of chemostat selective power, to attain these ends. Microarray chip analysis and intracellular redox sensors will be employed to determine how global gene expression and oxidative stress in P. putida are altered when the improved proteins are expressed, as well as the difference made by strain improvement to the bacterium’s response to the DOE waste. More detailed basic information on the relevant genes, evolution of more effective enzymes for chromate reduction, and P. putida strains with enhanced resistance to the DOE waste-site contaminants, as well as information base to devise rational approaches for enzyme and organismal improvement for specific bioremediation tasks will result. PROJECT: Elucidating the Molecular Basis and Regulation of Chromium(VI) Reduction by Shewanella oneidensis MR-1 and Resistance to Metal Toxicity Using Integrated Biochemical, Genomics and Proteomic Approaches PRINCIPAL INVESTIGATOR: Dorothea K. Thompson   Biomolecular Science and Engineering The mediation of metal reduction by microorganisms has been investigated intensively from physiological and biochemical perspectives; however, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform or immobilize a wide array of heavy metals contaminating DOE-relevant environments. Chromium(VI), for example, is one of several risk-driving contaminants at DOE sites and has been targeted by the DOE for bioremediation research. The bacterium Shewanella oneidensis MR-1 can potentially be used to immobilize chromium, a toxic and mutagenic metal, by reducing soluble Cr(VI) to the insoluble and less bioavailable form of Cr(III), thus facilitating its removal from contained-storage and natural sites. The overall goal of this study is to integrate targeted biochemical and proteomic analyses with microarray expression profiling to examine the molecular basis and regulation of chromium(VI) reduction by Shewanella oneidensis MR-1. Towards this goal, we will (1) isolate and identify the terminal chromium(VI) reductase and the gene(s) encoding this activity using whole-genome sequence information for MR-1 and mass spectrometry in conjunction with conventional protein purification and characterization techniques; (2) verify the function of the gene(s) encoding the terminal Cr(VI) reductase and compare whole transcriptome data with whole proteome data in order to understand the regulation of chromium reduction; and (3) examine chemotaxis and quorum sensing of S. oneidensis using a combination of protein biochemistry and molecular biology with regard to soluble Cr(VI). Use of whole-genome microarrays for S. oneidensis under conditions deemed toxic and attractive will help to describe the global cellular regulation of Cr(VI) reduction. This research will provide important information on the functional components and regulatory mechanisms of microbial metal reduction, which should prove valuable in developing effective assessment strategies for in situ bioremediation and genetically engineering desired bacteria for enhanced bioremediation.   [Back to Award Recipients Page]