1998 RESEARCH PROJECTS Program Element 3 Biomolecular Sciences and Engineering PROJECT: Metabolic Engineering of Marine Microorganisms for Heavy Metal Removal PRINCIPAL INVESTIGATOR: Douglas S. Clark PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: The primary objectives of the proposed work are to deter mine the mechanisms of metal chelation in a marine microorganism and to engineer (through molecular biological approaches) a superior metal-remediating microorganism. APPROACH: The specific objectives are: 1. To characterize the ability of strain 0420-1 to remove heavy metals (cadmium, lead, zinc) by forming insoluble sulfide complexes. 2. To determine the metabolic pathways for sulfide formation and efflux in strain 0420-1 and to overexpress these gene(s) in strain 0420-1 or Pseudomonas aeruginosa. 3. To engineer polyphosphate synthesis and degradation in a relevant environment. PROJECT: Design and Construction of Deinococcus radiodurans for Bioremediation of Radionuclides and Metals at DOE Waste Sites PRINCIPAL INVESTIGATOR: Michael J. Daly PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: The objective of this research is to develop novel organisms that are highly resistant to radiation and the toxic effects of metals and radionuclides. These organisms will be used for in situ bioremediation of radionuclides and metals, and to a lesser extent, organic toxins that could, theoretically, provide a carbon/energy source for metal immobilizing and mobilizing organisms. The soil bacterium Deinococcus radiodurans, the most radiation resistant organism discovered to date, will be the target for introducing test genes for metal remediation, as well as toxin degradation, alone or in combination. Metal remediating gene expression in D. radiodurans will be optimized using a variety of expression vectors and each novel strain will be characterized for metal transforming activity as well as for intermediate and terminal metabolites of organic toxin degradation. This research will provide radiation resistant bacteria with considerable potential for in situ and ex situ remediation of metals and radionuclides at high radiation, high toxicity DOE sites. APPROACH: This project has four specific research aims: Cloning and expressing genes from other organisms in D. radiodurans that can remediate radionuclides and metals, e.g., form Desulfovibrio and Shewanella, and degrade organic toxins, e.g., Pseudomonas. Assaying toxin-degrading gene expression in D. radiodurans in the presence and absence of radiation. Development of D. radiodurans strains capable of utilizing organic toxins as carbon and energy sources. Laboratory evaluation of engineered D. radiodurans strains for effectiveness at, e.g., reducing select radionuclides and metals in vitro and in natural subsurface materials from radionuclide/metal contaminated DOE sites containing organic co-contaminants. Such strains could be used in future research at DOE Field Research Centers. PROJECT: Stabilization of Radionuclides by Anaerobic Bacteria PRINCIPAL INVESTIGATOR: John J. Dunn PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: Clostridium sp. BC1 (ATCC No. 53464) (hereafter referred to as BC1) is an anaerobic, N2-fixing , gram-positive, rod-shaped bacterium isolated from coal-cleaning residues. This terrestrial, spore forming microorganism is a potential candidate for ameliorating radionuclide contamination at DOE sites as it has biochemical pathways that can convert water soluble uranyl ion U(VI), to less soluble U(IV). The initial objective of this project is to use a whole genome shotgun sequencing approach to obtain large amounts of BC1 DNA sequence information to discover key functional genes and to understand gene sequence/function relationships in this and related Clostridia/Bacillus species. Sequencing the BC1 genome will also provide an extensive base of knowledge regarding the physiology of this organism and aid in the identification of gene promoter sequences and their modes of regulation, ribosome binding sites, codon usage frequency and other features important for BC1 gene function. This information will eventually help facilitate expression in BC1 of engineered biodegradative pathways from other microorganisms to expand its usefulness as a tool for stabilization of radionuclides in different environments. APPROACH: Work is now in progress to sequence the ends of 2-3 kbp BC1 genomic DNA fragments cloned into pGEM vectors using vector-specific primers. Our initial goal is to obtain about 1.5 to 2-fold sequence coverage. At this stage more than 70% of the genes can be identified. BC1 clone libraries are also being constructed in a vector we have developed for generating sets of nested deletions whose ends are separated by ~400 bp. Fragments in the 10 kbp size range or larger can rapidly be sequenced using universal vector primers and any gaps remaining can be closed by primer walking on the original clone. The ability to sequence 10 kbp clones offers the potential for complete sequencing of interesting genes during the early phase of the project. We anticipate that most genes involved in intermediary metabolism, as well as the relevant reductases in reduction of U and N2, can be completely sequenced by this approach . Work is also in progress to construct a physical map of the AT-rich BC1 genome and to obtain a large insert (~35 kbp) fesmid clone library at 10x coverage to provide a network of genomic linking clones. PROJECT: Genes and Functions Regulated by adnA in Pseudomonas fluorescens PRINCIPAL INVESTIGATOR: Stuart B. Levy PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: The capacity of a bacterial cell to sense and respond to environmental changes is crucial to the cell's ability to adapt and survive in dynamic environments. The AdnA locus has been shown to be associated with a bacterial cell's ability to adhere to sands and seeds. Inactivation of this locus causes decreased survival and colonization of native soil. The gene shows strong peptide sequence homology to a subfamily of transcriptional activators that act as regulator members of two component sensor/regulator systems. Furthermore, sequences upstream of AdnA show similarity to the cognate sensor proteins of this subfamily. Because adhesion is a trait that is potentially important in cell survival, it is of interest to determine how the AdnA protein is involved in the transcriptional regulation of other loci that mediate the environmental success of the organism. A derivative of the AdnA locus whose expression is inducible by methylbenzoate will be constructed and introduced into the chromosome of Pseudomonas fluorescens strain Pf0-1. Genes regulated by AdnA will be cloned and sequenced and the DNA sequence will be used to search sequence databases for homology to previously characterized loci and to eventually clone and characterize new genes. Potential environmental signals that lead to transcriptional activation of loci by AdnA will be investigated. APPROACH: This research has four specific aims: 1) Construct a cassette containing the wild type AdnA gene under control of a methylbenzoate-inducible promoter. Introduce the inducible AdnA cassette into the chromosome of P. fluorescens wild-type strain Pf0-1 by gene replacement. 2) Use Pf0-1 containing the inducible AdnA cassette to identify AdnA-regulated chromosomal genes. Use random insertion mutagenesis of Pf0-1 with mini-Tn5 phoA and mini-Tn5-lacZ transposons and identify changes in gene expression associated with changes in the expression of AdnA. Determine observable changes in motility, adhesion to sand and seeds, antibiotic susceptibility, or other phenotypic changes resulting from the Tn5 (phoA or lacZ) inactivation of the gene. 3) Clone any AdnA-regulated genes using DNA adjacent to the mini-Tn5 insertion as a probe for the wild-type allele of the gene. Transfer the isolate wild-type allele of the gene into the insertion mutant to determine if there is reversion to the wild-type phenotype. Subclone the isolated DNA fragment to determine the smallest fragment capable of complementing the mutant phenotype. 4) Determine the DNA sequence of AdnA-regulated genes and conduct searches of sequence databases to determine if there is any similarity with previously described genes. PROJECT: Metal and Radionuclide Bioremediation by Starvation Promotor-Driven Combinatorial Bacteria PRINCIPAL INVESTIGATOR: Abdul Matin PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: Two problems complicate in situ bioremediation: a lack of nutrients and the presence of mixed wastes. A lack of nutrients hampers bioremediation because in wild type bacteria the expression of degradative activity is tightly coupled to rapid growth. A potential solution to this problem is the use of starvation promoters that are active in metabolically sluggish cells. APPROACH: This research will elucidate starvation and stress responses in Pseudomonas putida and apply the use of a starvation promoter for chromate remediation. A chromate reductase from P. ambigua will also be cloned and this and an already-isolated gene encoding a chromate efflux protein will be placed under the transcriptional control of a P. putida promoter that has already been identified. These constructs will be inserted into the chromosome of P. putida and the resulting strains will be tested by assaying for chromate reduction or chromate resistance. Non sterile "vadose zone material" will be added to both batch and c hemostat cultures to assess the ability of the engineered strains to compete with native organisms and maintain activity. The bioremedial activity of these strains will also be tested in soil columns using DOE site soils. In addition, the pathway of starvation sensing and signal transfer will also be determined by screening mutants for their inability to induce expression of a starvation promoter-lacZ fusion. The mutants and their cognate genes will then be cloned and sequenced. The network of starvation promoter induction will be determined by isolating extragenic suppressors of the starvation mutant, starv1, and cloning these genes. Finally, the half-life of chromate-related mRNAs and their translational efficiencies as expressed from starvation promoters will be determined. PROJECT: Complete Genome Sequencing of Shewanella putrefaciens PRINCIPAL INVESTIGATOR: J. Craig Venter PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: The objectives of the proposed work are to sequence the complete genome of Shewanella putrefaciens, to generate a databases of nucleotide and protein sequences and genome organization, and to make available a complete set of Shewanella genomic clones. APPROACH: The proposed research will consist of three phases: (1) Determine the complete DNA sequence of the Shewanella putrefaciens genome (approximately 4 Mb) (2) Edit, analyze, and annotate the Shewanella putrefaciens genomic sequence (3) Make the sequence data, annotation, and research materials, e.g., clones, available to the research community PROJECT: Genes for Uranium Bioremediation in the Anaerobic Sulfate-Reducing Bacteria PRINCIPAL INVESTIGATOR: Judy D. Wall PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: Reduction of U(VI) will be examined to determine the electron transfer components responsible. U(VI) reduction may occur by a fortuitous reaction. An interpretation that U(VI) reduction is fortuitous would be made if the electron transfer system required for U(VI) were also essential for reduction of an electron acceptor that would support growth. The conclusion that a dedicated uranium reductase function would be made if a mutant lacking the ability to reduce U(VI) were identified that has no other identifiable phenotype. If U(VI) reduction is mediated by an enzyme system necessary for reducing other oxidized species, such as Fe(III), Mn(IV), or O2 that do not support growth, the presence of an oxidation protection system will be explored. The presence of a global transcriptional regulator analogous to the E. coli Fnr will be explored. If present, the effects of such a global system on expression of the U(VI) reductase in Desulfovibrio will be analyzed. APPROACH: The specific objectives of the proposed research are: A. The identification of electron transfer components necessary for uranium reduction including: 1. Identification of a Desulfovibrio strain that can grow as colonies with a nonsulfidogenic substrate and can conjugate with E. coli plasmid donors. 2. Isolation of Desulfovibrio mutants that no longer reduce U(VI) and exploration of the reduction of other terminal electron acceptors by those mutants. 3. Isolation of mutants unable to reduce specific terminal electron acceptors normally used for growth and testing for their ability to reduce U(VI). 4. Isolation of mutants altered in pathways to other electron acceptors that are not known to support growth and testing for their ability to reduce U(VI). B. Determination of whether the sulfate reducing bacteria have and Fnr analog for the regulation of anaerobic terminal reductases. If so, the effects of mutations in the encoding gene on the ability of the cells to reduce U(VI) will be explored. PROJECT: Single Molecule Studies of Biodegradation of Radionuclide-Organic Complexes PRINCIPAL INVESTIGATOR: X. Sunney Xie PROGRAM ELEMENT 3 Biomolecular Science and Engineering OBJECTIVE: The high mobility of radionuclides complexed with chelating agents such as ethylenediaminetetraacetate (EDTA), nitrilotriacetate (NTA), and diethylenetriamine pentaacetate (DTPA) in groundwater represents a major environmental concern at DOE sites. Biodegradation of the chelating agents by microorganisms may help immobilize radionuclides in the environment. NTA monooxygenase from an NTA degrading microorganism and EDTA monooxygenase from an EDTA-degrading microorganism have been purified and characterized, providing an opportunity to understand the molecular basis for the biodegradation process. The objective of the project is to acquire molecular-level understanding of the enzymatic activities of these enzymes using a new single-molecule approach. 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. . APPROACH: Recent advances in single-molecule microscopy/spectroscopy, including those made at PNNL, have yielded detailed information regarding molecular interaction and chemical dynamics not obtainable from conventional experiments done on large ensembles of molecules. Our first hypothesis is that the activity of each enzyme molecule is different. The difference is only observable by single-molecule spectroscopy. Our second hypotheses is that the decrease in enzyme activity is due to the sudden loss rather than the gradual declination in activities of individual enzyme molecules. We will first establish a new approach for detailed characterization of enzymatic activity and stability by single-molecule measurements. We will also utilize the single-molecule approach to obtain detailed molecular-level understanding of enzymatic kinetics in the biodegradation systems. Using the single molecule enzyme essay, we will obtain more effective enzymatic systems by screening chemically and genetically modified NTA monooxygenase and EDTA monooxygenase. This interdisciplinary and collaborative effort may result in a new methodology, beneficial not only to selection, design, and refinement of bioremedial enzymes, but also to enzymology in general. [Back to Award Recipients Page]