2000 RESEARCH PROJECTS Program Element 3 Biomolecular Sciences and Engineering PROJECT: Engineering Deinococcus radiodurans for Metal Remediation in Radioactive Mixed Waste Environments PRINCIPAL INVESTIGATOR: Michael J. Daly, Ph.D. PROGRAM ELEMENT 3 Biomolecular Science and Engineering Seventy million cubic meters of ground and three trillion liters of groundwater have been contaminated by leaking radioactive waste generated in the United States during the Cold War. A cleanup technology is being developed based on the extremely radiation resistant bacterium Deinococcus radiodurans that is being engineered to express bioremediating functions. Research aimed at developing D. radiodurans for metal remediation in radioactive wastes sites was started by this group in September 1997 with support from DOE NABIR grant DE-FG02-97ER62492. Work funded by the existing grant has contributed to twelve papers on the fundamental biology of D. radiodurans and its design for bioremediation of radioactive waste environments - several of the research papers received extensive coverage by national and international news agencies. Our progress since September 1997 closely matches the Aims proposed in our previous NABIR application and is summarized as follows. We have demonstrated that D. radiodurans can be genetically engineered for metal remediation using four different expression systems, that were tested during growth of engineered D. radiodurans at 6,000 rad/hour. A variety of metal reducing/resistance functions have been cloned into D. radiodurans and are being studied, including genes from the following organisms that are specific for the indicated metal ions: Escherichia coli (merA), Hg(II); Desulfovibrio vulgaris (cytc3), U(VI); Ralstonia eutrophus CH34 (czc), Cd(II), Zn(II), and Co(II); and Bacillus thuringiensis, Cr(VI). Further, we have shown that anaerobic cultures of wildtype D. radiodurans can reduce U(VI) and Tc(VII) in the presence of humic acids; and Cr(VI) can be reduced in the absence of humic acids. Our development of a synthetic minimal medium for D. radiodurans was central to this work. This medium has also enabled us to identify the minimum nutrient requirements necessary to support growth in highly radioactive environments, and to explain how radiation resistance relates to this organism’s metabolic repertoire, predicted by analysis of its recently acquired genomic sequence. The availability of such a minimal medium, in which D. radiodurans’ growth is entirely dependent on a single carbon source, is also essential to our ongoing experimental efforts to survey its metabolic pathways and to engineer D. radiodurans for growth on toxic organic compounds present in most metal-contaminated radioactive waste sites. The D. radiodurans genomic sequence has been an important guide throughout this work and continues to be a source of inspiration in the development of new genetic technologies with which to understand and exploit this bacterium’s capabilities. Our planned NABIR work (2000-2003) is a natural extension of our existing collaborative NABIR project, outlining goals to further develop Deinococcus for metal remediation. The four specific goals are: 1) Cloning and expressing genes in D. radiodurans and Deinococcus geothermalis that can remediate radionuclides and metals, and to use and improve a novel technique to study gene expression patterns in D. radiodurans; 2) To study and expand D. radiodurans’ natural ability to reduce U(VI), Tc(VII), and Cr(VI); 3) Development of D. radiodurans strains capable of utilizing toluene and related compounds as carbon and energy sources during metal remediation - by metabolic pathway engineering; and 4) To test engineered D. radiodurans for effectiveness at metal remediation in natural subsurface materials. PROJECT: Metabolic Engineering of Microorganisms for Actinide and Heavy Metal Precipitation PRINCIPAL INVESTIGATOR: Jay D. Keasling PROGRAM ELEMENT 3 Biomolecular Science and Engineering Heavy metals and actinides are significant problems at a number of DOE sites and industrial locations in the U.S. Many of these sites contain heavy metals, actinides and organics. Due to the costs associated with excavating, transporting, and remediating contaminated sediments at remote locations, an economically viable solution is to mineralize the organic contaminants in situ and immobilize the metals and actinides to prevent movement to other locations. There are few reports in the literature of organisms capable of all of these functions. Besides their potential use in situ, these organisms should find use in treating wastes tanks at such sites as Hanford that contain mixed organics, metals, and actinides. During the previous grant period, we isolated and characterized a novel strain of Pseudomonas aeruginosa from a deep-sea hydrothermal vent capable of removing high levels of cadmium from solution by reducing thiosulfate to sulfide and precipitating cadmium as cadmium sulfide on the cell wall. To improve upon this system, we successfully engineered Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida to remove heavy metals and actinides from solution and immobilize them on the cell wall. For precipitation of cadmium, zinc, lead, and other metals that form strong sulfide complexes, we developed two systems for aerobic sulfide production: (1) expression of serine acetyl transferase and cysteine desulfhydrase in E. coli for overproduction of cysteine and subsequent conversion to sulfide; and (2) expression of thiosulfate reductase in E. coli and P. putida for reduction of thiosulfate to sulfide. The P. putida system was shown to allow simultaneous heavy metal precipitation and organics degradation. For precipitation of actinides as complexes of phosphate, we overexpressed polyphosphate kinase in E. coli and P. aeruginosa to enable these organisms to accumulate high levels of polyphosphate during phosphate excess and exopolyphosphatase for polyphosphate degradation and concomitant secretion of phosphate from the cell. All of these systems were shown to be capable of removing relatively high levels of metals from solution and have potential for metal and actinide removal from contaminated waste streams or immobilizing these elements in situ. In the next grant period we propose to engineer heavy metal and actinide precipitation in two microorganisms that will be relevant for treatment of DOE sites contaminated with heavy metals, actinides, and/or organics: Pseudomonas aeruginosa and Deinococcus radiodurans. Specifically, we propose: to engineer polyphosphate synthesis and degradation into Deinococcus radiodurans and P. putida for removal of uranium(VI) and plutonium(VI, V); to engineer aerobic sulfide production into D. radiodurans for removal of cadmium, zinc, and lead; to test removal of actinides; actinides and heavy metals; and actinides, heavy metals, and organics using the engineered organisms. PROJECT: Survival and Persistence Traits of Pseudomonas fluorescens PRINCIPAL INVESTIGATOR: Stuart B. Levy PROGRAM ELEMENT 3 Biomolecular Science and Engineering The survival of bacterial cells in the soil depends crucially on their ability to adapt to, and compete in, a constantly changing environment. The overall goals of this work are to identify the survival and persistence traits that allow soil bacteria to colonize a site. Several approaches will be taken. In the first, the regulation of motility and adhesion to sand and seeds will be studied. The adnA gene of Pseudomonas fluorescens is essential for motility and adhesion to surfaces, and is homologous to transcription activators of two component sensor/regulator signal transduction pathways. Inactivation of adnA causes decreased survival and colonization of native soil. Genes activated by AdnA will be identified by mutagenizing P. fluorescens with Tn5-lacZ or Tn5-phoA and comparing reporter activity of each mutant in the presence and absence of adnA expression. Other studies will examine the effects of environmental conditions on the expression of adnA and adnA-regulated genes. In a separate approach, alleles that confer a competitive advantage in minimal media or in sterile soil will be sought. Mutagenized P. fluorescens laboratory strains will be enriched for those variants best able to outcompete the parental strain. The alleles responsible for this phenotype will be sought using a combination of genetic complementation and analysis of changes in gene expression. Approaches that enhance survival of introduced bacteria will facilitate their in situ activity and may allow their long term establishment in the environment. PROJECT: Starvation Promoter-Driven Metal and Radionuclide Bioremediation in Combinatorial Bacteria PRINCIPAL INVESTIGATOR: Dr. A. C. Matin PROGRAM ELEMENT 3 Biomolecular Science and Engineering Environmental pollution by carcinogens is a serious problem. Chromate, for example, is a highly prevalent heavy metal contaminant specially at the Department of Energy sites. Using biomolecular approaches our aim is to (a) enhance bacterial ability to survive the harsh conditions of the contaminated environments;(b) to use expression elements (promoters) that are effectively switched on under stressful conditions; and(c) to improve biochemical effectiveness of proteins to detoxify the contaminants. We have recently found that chromate reducing activity is shared by several proteins that have been studied in very different contexts. These enzymes appear to possess low substrate specificity and share the property of delivering high-energy electrons. They may in fact be stress proteins with the prime function of neutralizing oxidizing agents. The genes encoding these enzymes share a high degree of homology. We are employing high resolution structural studies in conjunction with molecular approaches, such as DNA shuffling, to generate an enzyme species with greater effectiveness in remediating chromate and other high priority contaminants. PROJECT: Genes for Uranium Bioremediation in the Anaerobic Sulfate-Reducing Bacteria PRINCIPAL INVESTIGATOR: Judy D. Wall PROGRAM ELEMENT 3 Biomolecular Science and Engineering Cost effective bioremediation of radionuclides and metals in the subsurface will necessitate an understanding of the metabolic interactions of the anaerobic microorganisms that are found there. This knowledge will contribute to the elucidation of what is happening without intervention or what may happen with nutrient or microbial amendments. Among the bacteria inhabiting the subsurface are the sulfate-reducing bacteria, a heterogeneous group displaying a remarkable versatility in substrate utilization. Many members of this group have been shown capable of U(VI) reduction to U(IV), converting the uranium from a soluble species to a highly insoluble form that potentially could be removed from contaminated waters by precipitation or filtration. Genetic investigation into the pathway of reductant flow to U(VI) in bacteria of the genus Desulfovibrio will be continued. In D. desulfuricans strain G20, we have confirmed the importance of cytochrome c3 by disruption of the gene encoding that cytochrome, cycA, and demonstration of a decrease in the ability of the mutant to reduce U(VI). Our studies have also revealed an unexpected decrease in cytochrome c3 content when cells were grown in the presence of U(VI). Because this decrease results in attenuation of the reduction process, an elucidation of the phenomenon will be necessary to understand its prevalence and to design experiments to overcome this affect. Studies of the CycA mutant also revealed that multiple pathways for U(VI) are present in this strain. Biochemical and genetic approaches are being used to identify these alternate pathways. The isolation of the genes and determination of the regulation of their expression will reveal the physiological roles and assess their flexibility for manipulation. As a part of the regulation studies, the presence of global regulators responsible for differential expression of terminal electron acceptors will be sought. PROJECT: Single-Molecule and Biochemistry Studies of Flavin Channeling between Flavin Reductases and EDTA Monooxygenase PRINCIPAL INVESTIGATOR: Sunney Xie PROGRAM ELEMENT 3 Biomolecular Science and Engineering The high mobility of radionuclides complexed with chelating agents such as ethylenediaminetetraacetate (EDTA), nitrilotriacetate (NTA), and diethylenetriamine-pentaacetate (DTPA) represents a major environmental concern at DOE sites. Biodegradation of the chelating agents by microorganisms can help immobilize radionuclides in the environment. Recently, an NTA monooxygenase from an NTA degrading microorganism and EDTA monooxygenases from EDTA-degrading microorganisms have been purified and characterized, thus providing an opportunity to understand the molecular basis for the biodegradation process. Both NTA monooxygenase and EDTA monooxygenase belong to the group of FMNH2-utilizing monooxygenases that requires flavin reductases to supply FMNH2. All such enzymes reported to date have their own flavin reductase. FMNH2’s high reactivity prompts the question of whether the reductase delivers FMNH2 directly to the monooxygenase by metabolic channeling. The goal is construct genetically engineered microorganisms with these genes to destruct chelating agents and stop transport of heavy metals and radionuclides in the groundwater mediated by chelating agents. To do so, we need to know how the reductase and NTA or EDTA monooxygenase interact with each other. In this proposal, we describe our multidisciplinary efforts to study this interaction. The studies will combine the capability of single-molecule microscopy/spectroscopy developed by the Principal Investigator’s (PI) group with the expertise of the Co-Principal Investigator’s (Co-PI) group in the biochemistry and molecular biology of these enzymes. The single-molecule approach will be used to identify the channeling between flavin reductases and NTA and/or EDTA monooxygenase. The knowledge generated from the proposed studies will lead to a deeper understanding of the enzymatic reactions involved in chelating agent degradation and provide guidance for genetic modification of organisms and enzymes to improve their bioremedial activities. The proposed research will also create new enzymes that may have better catalytic activities for the degradation of chelating agents and stop chelating agents mediated transport of heavy metals and radionuclides in the subsurface environments. [Back to Award Recipients Page]