2002 RESEARCH PROJECTS
Program Element 3
Biomolecular Sciences and Engineering


PROJECT: Lateral gene transfer among subsurface bacteria
PRINCIPAL INVESTIGATOR: Tamar Barkay
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

The results of our current NABIR project show that metal resistances are widespread among chemoheterotrophic bacteria from the deep subsurface and that lateral gene transfer (LGT)* has contributed to the dissemination of these resistances. These findings raise three questions:

  1. What are the mechanisms of LGT among subsurface populations? To answer this question we will investigate the linkage between metal resistance genes and genetic elements that enhance LGT, and characterize molecular signatures that might have been generated by transfer events. We hypothesize that transposition and recombination events have facilitated LGT of metals resistance genes in subsurface strains.

  2. What is the diversity of plasmids and their compatibility determinants in bacteria from subsurface communities? To answer this question we will characterize plasmids and their inc/rep loci from subsurface strains. We hypothesize that novel lineages of plasmids confer metal resistance phenotypes in subsurface isolates.

  3. Has LGT contributed to metal resistance among strains of iron and sulfate reducing bacteria whose activities immobilize metals and radionuclides? To answer this question we will investigate metal resistance and LGT among sulfate and iron reducing bacteria from the deep subsurface. We hypothesize that although metal toxicity is reduced under anaerobic conditions, resistance facilitates survival and therefore promotes reduction of metals and radionuclides by subsurface strains.


Metal resistance may facilitate the survival of microorganisms in mixed waste sites and the spread of resistance by LGT may enhance the microbial induced immobilization of metals and radionuclides in the deep subsurface. The proposed study will expand our knowledge on genetic diversity among microorganisms in their natural habitats and on metal homeostasis among anaerobic microbes from the deep subsurface.

*Abbreviations used in this proporsal: BHR-broad host range; bp-base pair; DSMZ-German collection of microorganisms and cultures; FRC-Field Research center; GRE-gene recruiting elements; HgR-mercury resistance; HTP-high throughput; In-integron; Inc-incompetability; IS-insertion sequence; IRB-iron reducing bacteria; LGT-lateral gene transfer; NHR-narrow host range; ORF-open reading frame; RAPD-PCR-randomly amplified polymorphic DNA-PCR; RFLP-restriction enzyme length polymorphism; SRB-sulfate reducing bacteria; SMCC-subsurface microbial culture collection; SRS-Savannah river site; Tn-transposon.


PROJECT: Mechanism of uranium reduction by Shewanella Oneidensis
PRINCIPAL INVESTIGATOR: Thomas J. DiChristina
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

Microbial U(VI) reduction is an attractive alternative strategy for bioremediation of uranium-contaminated subsurface environments. Traditional ex situ remediation processes (e.g., ion exchange, biosorption or biomineralization) are often limited by poor extraction efficiency, inhibition by competing ions, production of large volumes of produced waste or uranium toxicity. Microbial U(VI) reduction provides an attractive alternative in situ remediation strategy since extracellularly produced U(IV) precipitates as uraninite (UO2), a highly immobile uranium mineral. Despite the potential benefits of enzymatic uranium precipitation, the molecular mechanism of microbial U(VI) reduction is poorly understood. The main goal of the DOE-NABIR project is to determine the molecular mechanism of anaerobic U(VI) reduction by Shewanella oneidensis. Complementary genetic and biochemical approaches will be used to clone the S. oneidensis U(VI) reduction genes and identify putative respiratory chain components, including the U(VI) terminal reductase. Genes encoding putative U(VI) terminal reductases will be cloned in an expression system and the resulting gene products purified. Purified U(VI) terminal reductases will be analyzed for signature structural motifs and electron donor oxidation and electron acceptor reduction activities. Purified U(VI) reductase will also be used as the antigen to develop U(VI) reductase antibodies for determining the subcellular location of the U(VI) reductase in S. oneidensis.

 

 

PROJECT: Construction of Whole Genome Microarrays for Desulfovibrio vulgaris and Expression Analysis of Cells Grown Under Uranium-Reducing Conditions
PRINCIPAL INVESTIGATOR: Matthew W. Fields
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

Microbial bioremediation of metals and radionuclides is a possible mechanism for the treatment of contaminated groundwater and sub-surface sediments. The genetic and physiological capabilities of microorganisms for the reduction and transformation of environmentally toxic metals and radionuclides are evident in nature and the laboratory, however, little is known regarding the molecular mechanisms, regulatory networks, and/or cellular responses controlling these processes. Access to the genomic contents of metal and radionuclide-reducing bacteria combined with microarray technologies provides an opportunity to elucidate metal/radionuclide respiratory pathways via whole genome expression analysis, and could provide avenues for predictable and effective bioremediation practices. Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized. The capacity of this bacterium to reduce different metals and radionuclides enzymatically has been demonstrated, and the focus of the proposed work is the identification and characterization of the cellular mechanisms for these reductions. Our primary goals are the: 1) construction of a whole-genome microarray for Desulfovibrio vulgaris and 2) use of the microarray to demonstrate cellular responses to uranium.


PROJECT: Mechanisms for the reduction of actinides and Tc(VII) in Geobacter sulfurreducens
PRINCIPAL INVESTIGATOR: Jon R. Lloyd
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

Uranium and technetium are the primary radioactive metals contaminating subsurface environments at Department of Energy (DOE) sites. Dissimilatory Fe(III)-reducing microorganisms can control the mobility of these contaminants through the enzymatic reduction of highly soluble U(VI) and Tc(VII) to insoluble tetravalent forms which will precipitate from groundwater and be immobilized in the subsurface. The mechanisms for the microbial reduction of U(VI) and Tc(VII) are relatively poorly understood, however, especially in the dissimilatory metal-reducing microorganisms that are likely to be most important in U(VI) and Tc(VII) reduction in the subsurface. It is vital, therefore, to know more about these mechanisms in order to rationally design in situ bioremediation strategies for uranium- and technetium-contaminated subsurface environments using dissimilatory metal-reducing microorganisms. The objective of the proposed research is to characterise the mechanisms of U(VI) and Tc(VII) reduction in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens. G. sulfurreducens has been chosen for study because: 1) this organism is closely related to the predominant organisms that emerge when dissimilatory metal reduction is stimulated in subsurface environments by the addition of various electron donors and/or electron shuttling compounds; 2) the genome sequence of this organism has been completed; and 3) a genetic system for this organism is available. In a previous 2-year NABIR-funded study we purified and characterized a periplasmic cytochrome c7 that reduced a range of electron acceptors in vitro, including U(VI), Fe(III) and humics. Confirmation that the protein was required for the reduction of these electron acceptors in vivo was obtained using a deletion mutant lacking the structural gene for the cytochrome. The mechanism of enzymatic Tc(VII) reduction was also characterized in whole cells. The oxidation of hydrogen was coupled to the reduction of Tc(VII) leading to the precipitation of Tc(IV) in the periplasm. The involvement of a periplasmic Ni/Fe hydrogenase was implicated by CO profiling. The aims of this proposed study are to use the tools of biochemistry and molecular biology to confirm the identity of the genes encoding the relevant U(VI) and Tc(VII) reductases in G. sulfurreducens and to elucidate the detailed mechanisms of U(VI) and Tc(VII) reduction by the corresponding enzymes. Furthermore, we propose to explore the range of other metals and radionuclides reduced by Geobacter sulfurreducens (including Np(V), Pu(IV) and Hg(II)), and identify the roles of the U(VI) and Tc(VII) reductases in the reduction of these other priority pollutants. The specific hypotheses that will guide our research are: (1) Cytochrome c7 functions as the U(VI) reductase of G. sulfurreducens in vivo, and is also capable of reducing and modifying the solubility of other actinide species including Np(V) and complexed Pu(IV), and toxic metals including Co(III) and Hg(II), via a mechanism that is distinct to that catalyzing the transfer of electrons to insoluble Fe(III) oxides. (2) Key amino acid residues can be mutated to identify regions of cytochrome c7 that are required for the reduction of metals and radionuclides. (3) A periplasmic Ni/Fe hydrogenase is the Tc(VII) reductase of G. sulfurreducens. This enzyme is also able to reduce a range of other electron acceptors, including U(VI), directly when hydrogen is supplied as the electron donor. (4) Additional genes may be required for U(VI) reduction in G. sulfurreducens, and can be identified by transposon mutagenesis.

This proposal builds on extensive experience of the PIs in microbial biochemistry and physiology (Lloyd), augmented with expertise in actinide chemistry (May and Livens). We will also continue successful collaborations with experts in protein crystallography (Dr Marianne Schiffer of The Argonne National Laboratory) and the molecular biology of Geobacter (Dr Derek Lovley of The University of Massachusetts).


PROJECT: Biomolecular Mechanisms for Microbe-Fe(III) Oxide Interactions in Geobacter species
PRINCIPAL INVESTIGATOR: Derek Lovley
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

The most promising strategy for the in situ bioremediation of radioactive groundwater contaminants that has been identified by the NABIR program is to stimulate the activity of dissimilatory metal-reducing microorganisms to reductively precipitate uranium, technetium, and radioactive cobalt. Previous studies with a variety of subsurface sediments, including those at uranium mine tailings (UMTRA) sites, have indicated that Geobacteraceae are the primary agents for metal reduction and that, even when uranium levels are high, electron transfer to Fe(III) reduction accounts for ca. 99% of the growth of the Geobacteraceae. These results suggest that in order to understand the factors controlling the growth and activity of the predominant U(VI)-reducing microorganisms during subsurface bioremediation it is important to understand how Geobacteraceae interact with the Fe(III) oxides. Preliminary studies have demonstrated that Geobacteraceae specifically produce pili and flagella when growing with insoluble Fe(III) oxide as the electron acceptor and that these appendages are important in aiding Geobacter species in accessing insoluble Fe(III) oxides. Therefore, the objective of this research is to investigate the outer surface of Geobacter species and to determine what outer surface structures these organisms use to access insoluble Fe(III) oxides. In the proposed research we will: 1) evaluate with novel proteomic approaches what proteins in Geobacter species, other than pili and flagella, are exposed to the extracellular environment; 2) determine which of these proteins are specifically expressed during growth on Fe(III) oxide; 3) determine with immunological techniques if, as hypothesized, these proteins are localized on one side of the cell; 4) examine the role of these proteins in cell-Fe(III) oxide interactions with genetic techniques and biological force microscopy; 5) determine the lipid composition of the cell membranes and potential changes in membrane composition during growth on Fe(III) oxide; and 6) use state-of –the-art electron microscopy procedures to examine the structure of the outer surface of the cell during growth on soluble electron acceptors and insoluble Fe(III) oxide.

These studies combine expertise in the physiology of Geobacteraceae (UMASS) with expertise in the analysis of microbial surface structure and cell-metal interactions (U. Guelph) as well as expertise in novel proteomics approaches (PNNL). This research is expected to provide insights into the factors controlling the growth and metabolism of Geobacteraceae during in situ bioremediation of uranium and to identify molecular targets that can be used to assess the activity of Geobacteraceae in the subsurface.



PROJECT: Engineering MerR for Sequestration and MerA for Reduction of Toxic Metals and Radionuclides
PRINCIPAL INVESTIGATOR: Anne Summers
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

Our aim is to use genetic engineering to elicit biochemical novelty and diversity and to tailor this diversity to solve metal and radionuclide remediation problems. This is a technique-intensive proposal, but does include attention to key basic science issues. Given the relative paucity of high throughput screening and selection tools available for metallobiology, technique development is essential for advancing both basic and applied science in this major area of biology.
We will test the hypotheses that:

  1. The well-characterized metal binding protein, MerR, and the flavin metal reductase, MerA, can be engineered to be effective at binding and reduction (respectively) of metals of interest to DOE, including chromium, technitium, and uranium.

  2. Such engineered proteins, expressed in the same bacterial strain, will protect bacteria from the toxic effects of the metals while allowing them to reduce the metals to a form more readily recoverable ex situ.

  3. The basic science insights derived from these engineering exercises will illuminate the natural evolution of proteins for metal binding and as metal redox catalysts.

To these ends we propose specifically to:

  1. Determine the nature of Cr, Tc and U binding by MerR and our recently constructed single- polypeptide tandem Metal Binding Domain (MBD) protein;

  2. Devise methods for high-throughput screening and selection of MerR variants which bind Cr, Tc, or U and MerA variants which reduce Cr or U.

  3. Employ both targeted and random mutagenesis and select or screen for variants with novel binding (MerR) and redox (MerA) specificities;

  4. Employ novel MerR variants with MerA variants for in vivo metal sequestration in small cultures and also in pilot scale fermenters.

 

PROJECT: The Role and Regulation of Melanin Production by Shewanella oneidensis MR-1 in Relation to Metal and Radionuclide Reduction and Immobilization
PRINCIPAL INVESTIGATOR: Charles E. Turick
PROGRAM ELEMENT 3 Biomolecular Science and Engineering

Bacteria in the genus Shewanella grow by using soluble and insoluble metals for energy production. Humic compounds are known to accelerate the process by which microorganisms transfer electrons to metals, thereby decreasing metal toxicity and mobility. The pigment melanin is a particularly important humic compound in this process, and is produced by bacteria in the genus Shewanella. In the presence of melanin S. algae BrY reduces the insoluble mineral hydrous ferric oxide as much as ten times faster than without melanin. This is accomplished because, under anaerobic conditions, melanin serves as a terminal electron acceptor and soluble electron shuttle to iron minerals.


The overall hypothesis of this work is this: Melanin production in the genus Shewanella plays a significant role as a mechanism of metal and radionuclide reduction and immobilization, and its production can be manipulated with the addition of proper nutrients. By understanding the role and regulation of melanin production in microorganisms, remediation of metal and radionuclide-contaminated environments may be accelerated.


This work will focus on the role of melanin production by the bacterium S. oneidensis MR-1 in relation to a mechanism for metal and radionuclide reduction. The significance of melanin’s role in metal reduction will be determined by preventing melanin production by inhibiting the enzyme responsible for melanin production as well as the generation and use of mutants deficient in melanin production. S. oneidensis cultures will be evaluated for their capacities for metal reduction relative to melanin production.

 

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