Biomolecular Sciences and Engineering

PROJECT: Molecular Mechanisms of Uranium Reduction by Clostridia and its Manipulation

A.J. Francis

Biomolecular Science and Engineering

Subsurface contamination by radionuclides and toxic metals is a major problem across the DOE complex. Removal of contaminated media is financially prohibitive. Consequently, innovative, cost effective, in situ stabilization technology by exploiting the natural attenuation process must be developed. Microbial stabilization of actinides (U,Pu, Np) and fission products (Tc) in subsurface environments is currently being investigated at DOE sites. A wide variety of bacteria including the strict anaerobic spore forming Clostridia are involved in the reductive precipitation of uranium and Tc in subsurface environments. Although the mechanisms of U reduction by dissimilatory metal-reducing bacteria (DMRB) Geobacter and Shewanella, and sulfate-reducing bacteria (SRB) Desulfovibrio, have been extensively investigated, little is known of the mechanisms of uranium reduction by fermentative bacteria such as Clostridia. It is postulated that the excess electrons generated during fermentation of organic materials are used in the uranium reduction process. This research addresses the need for detailed studies of the enzymatic mechanisms for the reduction of radionuclides and/or metals by fermentative microorganisms. The overall objective of this research is to elucidate systematically the molecular mechanisms involved in the reduction of uranium by Clostridia. We propose to (i) determine the role of hydrogenases in the reduction of uranium, (ii) purify the enzymes involved in uranium reduction, (iii) determine the mechanisms of reduction e.g., one or two electron transfer reactions, and (iv) elucidate the genetic control of the enzymes and cellular factors involved in uranium reduction. Speciation and intermediate oxidation states of uranium will be determined electrochemically and by X-ray adsorption near edge structure (XANES) at the National Synchrotron Light Source (NSLS). Fundamental knowledge of a molecular assessment of radionuclide and metal reduction will allow us to exploit the naturally occurring processes to attenuate radionuclide and metal contaminants in subsurface environments dominated by low and high pH, high nitrate, and/or organic matter where dissimilatory metal reducing bacterial activity will be limited. This is a collaborative study between BNL and Stanford University involving expertise in biomolecular science, biochemistry, microbiology and electrochemistry.

PROJECT: Biomolecular Mechanisms Controlling Metal and Radionuclide Transformations in Anaeromyxobacter dehalogenans
Biomolecular Science and Engineering

Microbiological reduction of U(VI) and Tc(VII) has been proposed as a strategy for remediating radionuclide-contaminated environments. Numerous studies focusing on the reduction kinetics and speciation of these metals have been carried out using contaminated sediment samples, microbial consortia, and pure bacterial cultures. While previous work with model organisms has increased the general understanding of radionuclide transformation processes, fundamental questions regarding radionuclide reduction mechanisms by indigenous microorganisms are poorly understood, especially under the commonly encountered scenario where multiple electron acceptors are present. Therefore, the overall goal of the proposed research is to elucidate the molecular mechanisms of radionuclide biotransformation by Anaeromyxobacter  dehalogenans, and to assess the effects of relevant environmental factors on these transformation reactions. Members of the Anaeromyxobacter genus have been found in a range of undisturbed and contaminated soils and sediments. Importantly, Anaeromyxobacter species were shown to occur in the U(VI)-contaminated, acidic sediments of the Field Research Center (Oak Ridge, TN). The proposed research will integrate targeted physiological and genetic analyses with microarray expression and genotype profiling to elucidate the mechanisms of metal transformation reactions in an environmentally relevant bacterial group. Further we will determine the effects of co-contaminants (e.g. nitrate, chlorinated solvents) on radionuclide reduction. Established chemostat cultivation techniques will be used to produce cells under precisely controlled and defined conditions. The distribution and diversity of genes involved in metal and radionuclide reduction among different A. dehalogenans strains, including those detected in enrichment cultures derived from FRC site material, will be assessed using a microarray-based comparative genomics approach. This research effort will generate novel understanding of the mechanisms involved in metal reduction and enhance our predictive capability of the processes that govern radionuclide transformation reactions in subsurface environments. Ultimately, these findings will assist the design and implementation of more efficient bioremediation approaches to enhance the reductive transformation and immobilization of radionuclides at contaminated DOE sites.

PROJECT: Nanowires, Capacitors, and Other Novel Outer-Surface Components Involved in Electron Transfer to Fe(III) Oxides in Geobacter Species
  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 as well as the toxic metal vanadium. Previous molecular studies of a variety of subsurface environments, including several uranium-contaminated DOE sites, have clearly indicated that Geobacteraceae are the primary agents for metal reduction and that, even when uranium levels are high, electron transfer to insoluble Fe(III) oxides accounts for ca. 99% of the growth of the Geobacteraceae. When Fe(III) oxides are depleted the growth and activity of Geobacteraceae stop and U(VI) is no longer reduced. These results demonstrate that in order to design strategies to optimize in situ bioremediation of metals it is imperative to understand how Geobacteraceae transfer electrons to insoluble Fe(III) oxides. In this renewal we will follow up on our recent discovery that the pili of Geobacter sulfurreducens are conductive and appear to carry out the final electron transfer onto Fe(III) oxides. We will also further examine the role of c-type cytochromes and other novel outer-membrane proteins that are specifically required for Fe(III) oxide reduction. The following hypotheses will be evaluated: 1) the pili of G. sulfurreducens are conductive along the complete length of the pili; 2) conductance of the pili can be attributed to one of several mechanisms for charge transport within the pilin molecular structure, or alternatively, within an electrolytic core inside the pilus; 3) other outer membrane proteins do not contribute to the conductance of G. sulfurreducens pili; 4) the conductance of pili might be attributed to metals binding on the pilin surface; 5) the pili of other Geobacteraceae are conductive in a manner similar to that previously observed in G. sulfurreducens; 6) the pili of other dissimilatory Bacteria and Archaea that need to directly contact Fe(III) oxides in order to reduce them are also conductive; 7) some of the other electron transport proteins that are involved in electron transfer to Fe(III) oxide and function as intermediaries in the final electron transfer to pili are, like pili, localized on one side of the cell; 8) the abundant c-type cytochromes found in Geobacter species can function as capacitors, permitting temporary energy conservation via electron transfer in the absence of an external electron acceptor; and 9) other outer-membrane proteins that are highly conserved in the Geobacteraceae, but not found in other organisms, play a role in Fe(III) oxide reduction. These hypotheses will be investigated with a combination of microbiological, genetic, and electrochemical methods that have been well-developed in the first two years of these studies. This research will provide insights necessary for predictively modeling in situ bioremediation of uranium and other toxic metals and will identify molecular targets that can be used to assess the activity of Geobacteraceae during in situ bioremediation.


PROJECT: Integrating the Molecular Machines of Mercury Detoxification into Host Cell Biology
  Biomolecular Science and Engineering

The bacterial mercury resistance (mer) operon, one of the most evolutionarily successful genetic loci in any defined organism, detoxifies organic and inorganic mercury compounds. Several major biotic processes in the global Hg(II) cycle are carried out by bacteria with this highly mobile detoxification locus that occurs in Gram negative and high and low GC Gram positive bacteria. The functions of many individual mer operon components are well described, so we aim to dissect the higher order interactions of the enzymes, transporters, and regulators of this paradigm metal metabolizing system with each other and with the larger metabolism of the host cell. Understanding how this ubiquitous detoxification system fits into the biology and ecology of its bacterial host is essential to guide interventions that support and enhance Hg remediation. Specifically, we will test the hypotheses that: (a) the organomercurial lyase, MerB, and the mercuric reductase, MerA, act synergistically together and with the membrane-bound Hg(II) transporters, MerT and MerC, to detoxify mercurials; (b) the interaction of the metalloregulator MerR with RNA polymerase (RNAP) and with its DNA binding site, MerO, modulates its metal response, and interaction with its antagonist, MerD, prevents RNA polymerase from binding to the structural gene promoter, P merT and (c) exposure of cells to Hg(II) makes specific demands on cellular resources and expression of the mer operon modulates those demands and is, in turn, modulated by them. To test these hypotheses we propose to: (a) use enzymology, NMR, fluorescence anisotropy, protein-crosslinking, crystallography, and calorimetry in vitro along with in vivo measurements of Hg(II) volatilization and HgR phenotyping to detect and define interactions between the mer enzymes, MerA and MerB, and the transporters, MerT and MerC,and their functional fragments and specific mutant variants; (b) use NMR, fluorescence anisotropy, protein-crosslinking, crystallography, affinity pull-downs, and calorimetry in vitro along with in vivo measurements of transcript synthesis and HgR phenotyping to detect and define interactions between mer regulatory proteins (MerR and MerD), DNA sites (MerO, PT and PR), and RNA polymerase and their functional fragments and specific mutant variants; and (c) use 2D microarrays to define the Hg-inducible transcriptome of the model bacterium E. coli and of radionuclideremediation model microorganisms, Shewanella oneidensis and Desulfovibrio vulgaris,with and without the mer operon. The information and insights obtained from this work will benefit the DOE-NABIR program by providing (a) a model for an evolutionarily successful metal detoxification system and (b) guidance for manipulations of field conditions so as to optimize the functioning of the cells which carry this detoxification system. The work will also contribute to the fundamental understanding of (a) the evolution of modular architecture in multi-domain proteins and (b) the integration of horizontally transferred genetic elements into pre-existing networks of cellular functions.


PROJECT: Molecular Mechanism of Microbial Technetium Reduction
  Biomolecular Science and Engineering

Microbial Tc(VII) reduction is an attractive alternative strategy for bioremediation of technetium-contaminated subsurface environments. Traditional ex situ remediation processes (e.g., adsorption or ion exchange) are often limited by poor extraction efficiency, inhibition by competing ions and production of large volumes of produced waste. Microbial Tc(VII) reduction provides an attractive alternative in situ remediation strategy since the reduced end-product Tc(IV) precipitates as TcO2, a highly insoluble hydrous oxide. Despite its potential benefits, the molecular mechanism of microbial Tc(VII) reduction remains poorly understood. The main goal of the proposed DOE-NABIR research project is to determine the molecular mechanism of microbial Tc(VII) reduction.Random mutagenesis studies in our lab have resulted in generation of a set of six Tc(VII) reduction-deficient mutants of Shewanella oneidensis. The anaerobic respiratory deficiencies of each Tc(VII) reduction-deficient mutant were determined by anaerobic growth on various combinations of three electron donors and 14 terminal electron acceptors. Results indicated that the electron transport pathways to Tc(VII), NO3-, Mn(III) and U(VI) share common structural or regulatory components. In addition, we have recently found that wild-type Shewanella are also able to reduce Tc(IV) as electron acceptor, producing Tc(III) as an end-product. The recent genome sequencing of a variety of technetium-reducing bacteria and the anticipated release of several additional genome sequences in the coming year, provides us with an unprecedented opportunity to determine the mechanism of microbial technetium reduction across species and genus lines. Our proposed research on the molecular mechanism of microbial technetium reduction is driven by three main hypotheses:

HYPOTHESIS NO. 1. The electron transport pathways of technetium-reducing bacteria share common structural or regulatory components.

HYPOTHESIS NO. 2. Technetium-reducing bacteria reduce Tc(VII) to Tc(III) step-wise via two successive electron transfer reactions catalyzed by separate Tc(VII) and Tc(IV) reductases.

HYPOTHESIS NO. 3. Tc(VII) and Tc(IV) reductase protein complexes may identified by combining a new proteomics system with MALDI-TOF mass spectrometry and peptide mass fingerprinting.

Traditional genetic complementation approaches will be used to clone genes required for Tc(VII) and Tc(IV) reduction in S. oneidensis. Targeted gene deletion mutagenesis will be followed to confirm involvement of homologous proteins in technetium reduction by other members of the genus Shewanella. Complementary bioinformatic analyses will be used to identify putative homologs in technetium-reducing bacteria of the genera Geobacter, Desulfovibrio, Escherichia and Deinococcus. A genome-enabled, proteomics approach will be used to identify technetium reductase protein complexes in all technetium-reducing bacteria whose genomes have been sequenced, regardless of phylogenetic affiliation.


PROJECT: Identification of Molecular and Cellular Responses of Desulfovibrio vulgaris Biofilms under Culture Conditions Relevant to Field Conditions for Bioreduction of Heavy Metals
  Biomolecular Science and Engineering

The use of planktonic growth conditions, in which cells exist as ‘non-adhered cells’, rarely represents a true state of growth for the majority of microorganisms under in situ conditions. It is becoming increasingly clear that a mode of attached growth more closely resembles in situ conditions for many microorganisms in different environments. Work by numerous researchers in the last five years has shown that different sulfate-reducing bacteria (SRB) can form biofilms. However, relatively little work has been done on anaerobic biofilms, particularly the structure and rheology of SRB biofilms when compared to the work performed with aerobic biofilms. Recent work with different Desulfovibrio spp. has shown that biofilms are formed by these SRB, and that the properties of sulfate- and metal-reduction are different compared to the growth of cell suspensions. However, the cellular processes of SRB biofilms in metal-reducing conditions have not been studied with genomic and proteomic approaches. It is crucial to characterize the overall cellular responses that constitute the processes responsible for heavy metal reduction and long-term immobilization in order to better design bioremediation strategies. Our primary goals for this project include 1.) transcriptomic and proteomic characterization of D. vulgaris biofilms grown under metal-reducing conditions, 2.) identification of key genes necessary for biofilm formation and maintenance, 3.) determination of expression patterns of biofilms cultivated in the presence of heavy metals, and 4.) characterization of the responses of D. vulgaris biofilms in the presence of mixed contaminants. To accomplish these objectives, we will use transcriptomic, proteomic, genetic, physiological, and microscropic analyses to characterize D. vulgaris biofilms cultivated in biofilm reactors by testing the following hypotheses:

Hypothesis 1: D. vulgaris biofilms have altered expression patterns and activities compared to nonadhered cells.

Hypothesis 2: Different subsets of genes will be expressed as D. vulgaris biofilms mature.

Hypothesis 3: D. vulgaris has key genes that are essential and/or necessary for significant biofilm formation under sulfate- and metal-reducing conditions.

Hypothesis 4: D. vulgaris biofilms are more resistant to heavy metals compared to cell suspensions.

Hypothesis 5: D. vulgaris biofilms are more resistant to mixed wastes compared to non-adhered cells.


PROJECT: Molecular Mechanism of Bacterial Attachment to Fe(III)-oxide Surfaces
  Biomolecular Science and Engineering

Attachment of dissimilatory iron reducing bacteria to Fe(III)-oxide and oxyhydroxide mineral surfaces has important implications for environmental Fe-cycling and water quality in subsurface environments, including the mobility and availability of metal and radionuclide contaminants. Cell attachment at Fe(III) mineral surfaces should facilitate the direct transfer of electrons from cell to mineral, an important process in subsurface environments with low dissolved organic carbon since extrinsic electron shuttling capacity is likely to be low. Attached, metabolically active cells represent a significant component in subsurface bioremediation processes in such systems. Obversely, cell attachment to surfaces in the vadose and subsurface environments can significantly limit cell transport through porous media, limiting the effective area that can be treated by bioaugmentation strategies where cells of a desired metabolic capacity are injected in contaminated sediments via a well head. We propose that gaining greater insight into the biomolecular basis for cell attachment to iron oxide minerals is essential, not only in understanding the predominant mechanism for direct electron transfer in subsurface environmentslacking in sufficient extrinsic electron shuttles (i.e. humic and fulvic acids), but also in predicting the transport of bacterial cells through porous vadose and subsurface environments. Our genomeenabled strategy includes generation of direct and random adhesion (Adh) mutant strains of Shewanella oneidensis MR-1. Adh mutants will be identified using a rapid adhesion bioassay in columns of Fe(III)-coated quartz sand. Identified Adh mutants, together with the wild-type strains, will be characterised physicochemically with a comprehensive suite of spectroscopic and physical measures to describe the phenotypes of Adh mutants compared to the wild-type strains. Measurement of cell zeta potential and surface tension will allow modeling of cell-mineral surface interactions employing extended DLVO theory, establishing the effects of phenotypic differences upon the physicochemical interaction at the cell-mineral interface. These models of cell-mineral surface interactions will be validated with whole cell adhesion assays, employing atomic force microscope-based and laminar flow reactor experiments performed with model iron oxide minerals surfaces and a Fe-free mineral analog. These bioassays will help identify specific interactions with iron at mineral surfaces. Having identified genes important in the adhesion of S. oneidensis MR-1 to iron oxide surfaces, we intend to apply the knowledge gained to investigate the distribution of the identified genes in other Shewanella species using the four Shewanella genome sequences currently available from the DOE Microbial Genome Program, and the expected additional genome sequences likely for release during the lifetime of our proposed work. If the S. oneidensis genes involved in Fe(III) oxide attachment are detected in a broad range of other Shewanella genomes, we will perform targeted gene deletion mutagenesis of those genes in the other Shewanella strains, and test the resulting deletion mutants for efficiency of attachment to Fe(III) oxide surfaces. These experiments will be used to test the hypothesis that all Fe(III)-reducing Shewanella attach to Fe(III) oxides via a common mechanism. Subsequently, in silico bioinformatic analyses will be carried out on bacterial genomes outside the genus Shewanella (e.g., Geobacter sulfurreducens, G. metallireducens and Deinococcus radiodurans) to determine if homologs of the previously identified Shewanella adhesion-related proteins are found in all Fe(III)-reducing bacteria, an indication that Fe(III)-reducing bacteria attach to Fe(III) oxides via a common mechanism.

Hypotheses - The following hypotheses address the biomolecular basis for dissimilatorymetal reducing bacterial interaction with mineral surfaces acting as electron acceptors: (1) Fe(III) and Mn(IV) reduction pathways of S. oneidensis involve cell envelopestructures or appendages that specifically adhere to solid Fe(III) and Mn(IV) oxidesurfaces and (2) Phenotypes displaying reduced attachment behavior will manifestdifferent surface physicochemistry, which can be identified either by spectroscopic investigation of the cell outer membrane, or by whole cell measures of cell surface zetapotential, surface free energy and hydrophobicity.

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