Michael J. Daly Lab: Deinococcus radiodurans

Funded Application for Financial Assistance

From the Office of Science U.S. Department of Energy, Office of Science, Grants and Contracts Division, SC-64, 19901 Germantown Road, Germantown, MD 20874-1290 ATTN: Program Notice DE-FG01-04ER04-06

Integrative Studies element of NABIR, combining the Biomolecular Sciences and Engineering, and Community Dynamics and Microbial Ecology elements

Characterizing the Catalytic Potential of Deinococcus, Arthrobacter and other Robust Bacteria in Contaminated Subsurface Environments of the Hanford Site

Principal Investigator

Michael J. Daly, Ph.D., Principal Investigator
Associate Professor
Uniformed Services University of the Health Sciences

Co-Investigators

James K. Fredrickson, Ph.D., Staff Scientist
Environmental Microbiology Group
Pacific Northwest National Laboratory


Abstract

Immense volumes of soil and groundwater at numerous U. S. Department of Energy (DOE) sites have low levels of widespread contamination that include mixtures of heavy metals (e.g., Hg & Cr) and radionuclides (e.g., U & Tc). The remediation of such contaminated sites constitutes an immediate and complex waste management challenge for DOE, particularly in light of the costliness and limited efficacy of current physical and chemical strategies for mixed wastes. In situ bioremediation via natural microbial processes (e.g., metal reduction) remains a potent, potentially cost-effective approach to the reductive immobilization or detoxification of environmental contaminants.

Several key findings for bacteria belonging to the families Deinococcaceae and Arthrobacteriaceae support their suitability as subjects for the Integrative Study element of NABIR, combining the elements Biomolecular Sciences and Engineering, and Community Dynamics and Microbial Ecology. We have recently isolated several distinct species of Deinococcus and Arthrobacter from pristine and contaminated soils of DOE's Hanford Site in south-central Washington state. Generally, Deinococcus bacteria are exceptionally robust, not only surviving exposure to radiation, but also to other DNA damaging conditions typical of DOE environments. The ability to reduce a variety of toxic metals also appears to be a characteristic shared by these organisms, and we have shown that Deinococcus radiodurans and Deinococcus geothermalis are proficient at reducing U(VI), Tc(VII) (in the presence of an electron shuttle) and Cr(VI), and have engineered both species for Hg(II) reduction. Recent experimental advances in the genetic management of D. radiodurans and D. geothermalis will facilitate our efforts to characterize the metal-reducing mechanisms of both organisms. These advances include our comprehensive analysis of the D. radiodurans genome, and for D. geothermalis, the development of a system for genetic transformation and ongoing genome sequencing, annotation, and analysis of this organism. Arthrobacter species are capable of free-living growth in many extreme environments and have also recently been reported to efficiently resist and reduce high concentrations of hexavalent chromium. These and other cultured isolates from the Hanford Site will be examined for their metal-reducing capabilities as outlined below.

The three investigators of this NABIR application have been working together on Deinococcus research since 1997 and now are also the Principal Investigators, respectively, of three independent DOE Genomes to Life (GtL) grants: Daly (Deinococcus), Fredrickson (Shewanella oneidensis), and Wackett (Database development for bioremediating genes). Additionally, Dr. Wackett also now leads the ongoing, NSF-sponsored, whole genome-sequencing project for Arthrobacter aurescens (TC-1). This application, therefore, represents a flexible collaborative expertise that will further contribute to defining and expanding integrative research efforts within DOE's NABIR program. Building on our systematic approach over the last seven years, the four specific aims of this proposal are: 1) Characterizing the genetic basis of metal reduction in D. radiodurans and D. geothermalis, and novel deinococcal isolates from the Hanford Site; 2) Characterizing the microbial ecology of contaminated waste sites and metal-reducing potential of these communities; 3) Defining environmental factors that govern in situ growth, environmental robustness, metabolism, and Cr(VI)-reduction of Deinococcus and Arthrobacter spp.; and 4) Characterizing the genetic basis of metal reduction in Arthrobacter aurescens (TC1) and novel Arthrobacter isolates from the Hanford Site.

Project Description

Characterizing the Catalytic Potential of Deinococcus, Arthrobacter and other Robust Bacteria in Contaminated Subsurface Environments of the Hanford Site

A. General and Specific Aims

The proposed research will build on a wealth of results generated by our research groups since 1997. Our ultimate aim is to identify organisms and approaches to remediate soil and groundwater at DOE sites that have low levels of widespread contamination of Cr, Hg, Tc, and U in subsurface environments below the zone of root influence that includes both the vadose zone and the saturated zone. Microbiological techniques suitable for metal reduction/immobilization could prevent or minimize dissemination of these contaminants. For bioremediation to be effective in such areas, bacteria must be able to withstand cellular toxicity caused by heavy metals and other environmental stress factors associated with waste environments. Additionally, they must detoxify the target contaminants without increasing the mobility or toxicity of co-contaminants. Since no single known organism has met all these requirements our emphasis will be on examining the catalytic potential of microbial communities from contaminated environments at the Hanford Site, which have recently become accessible for study through site characterization activities.

This application is being submitted under the Integrative Studies element of the NABIR program. Although the current NABIR solicitation does not include the Biomolecular Sciences and Engineering element, DOE has advised us that the Biomolecular Sciences element is not precluded under the Integrated Studies element. We have refocused our research efforts away from engineering Deinococcus strains to studies dedicated to the molecular characterization of two bacterial groups isolated from contaminated sediments at the Hanford Site in south-central Washington state. Of the bacteria isolated at the Hanford Site, Arthrobacter spp. are the most prevalent group and Deinococcus spp. are the most radiation resistant, both express naturally-encoded metal-reducing functions. We have aligned our aims to the elements 'Biomolecular Sciences/Engineering', and 'Community Dynamics/Microbial Ecology.' These complimentary approaches will allow us to better predict the composition of indigenous microbial communities, characterize their catalytic potential for bioremediation, and optimize expression of their metal-immobilizing functions. This application supports the fundamental science that will serve as the basis for development of cost-effective bioremediation and long-term stewardship of radionuclides and metals in the subsurface within these contaminated areas.

We propose to characterize the metal-reducing mechanisms of Deinococcus and Arthrobacter species, and other robust bacteria native to the Hanford Site. To achieve this goal, we have four specific research aims; these aims are summarized below and addressed in the Research Plan. For an overview of the Aims, the reader is directed to the flowchart of integrated Aims.

Aims

  1. Element: Biomolecular Sciences/Engineering. Characterizing the genetic basis of metal reduction in D. radiodurans and D. geothermalis, and novel deinococcal isolates from the Hanford Site. We will focus on characterizing Cr(VI) reduction and determine the substrate specificity (Cr(VI), Mn(IV), Fe(III), U(VI), Tc(VII)) of the enzyme complex(es). For example, Cr(VI)- and Mn(IV)-reduction functions might be related; both are environmentally relevant to the prevalent aerobic Hanford Site subsurface sediments since reduction of Cr(VI) yields relatively non-toxic and poorly soluble Cr(III), and assimilatory reduction of environmental Mn(IV) to Mn(II) is believed to be key to acquiring the high intracellular concentrations of Mn(II) necessary for growth of Deinococcus and radiation resistance. For D. radiodurans, the tryptic peptides from the purified Cr(VI) reductase proteins will be determined using mass spectrometry for identification, and the corresponding ORFs encoding these proteins in D. radiodurans and D. geothermalis will be disrupted by insertional inactivation using existing Deinococcus gene knockout protocols and subjected to phenotypic characterization.
  2. Element: Community Dynamics/Microbial Ecology. Characterizing the microbial ecology of contaminated waste sites and metal-reducing potential of these communities. We have characterized the distribution of viable microorganisms in contaminated and pristine vadose sediment samples from select locations at the Hanford Site including a contaminant plume in the vadose region under tank SX-108. Our approach to determining the phylogeny of cultivated and uncultivated members present in SX-108 sediments could also be applied to microbial communities within other subsurface sediments that will be collected at the Hanford Site in the near future as part of site characterization activities. The >110 cultured isolates from under tank SX-108, and those from non-contaminated sediments, were dominated by Gram-positive aerobic chemoheterotrophs. Representative isolates will be examined for their Cr(VI)-, Hg(II)-, and Mn(IV)-reducing capabilities with emphasis on Arthrobacter spp., the most abundant group of bacteria identified in unsaturated vadose sediments.
  3. Element: Community Dynamics/Microbial Ecology. Defining environmental factors that govern growth, environmental robustness, metabolism, and metal-reduction of Deinococcus and Arthrobacter spp. For Deinococcus, factors to be evaluated by a combination of (i) physiologic and (ii) microarray approaches will include: (i) examining D. radiodurans reductase mutants for their radiation/desiccation resistance, dependence on Mn(II), substrate utilization patterns, and the effect of O2 concentration; and (ii) differential expression profiling of wild-type and reductase mutants exposed to these physiologic conditions. Other environmentally robust Hanford Site isolates, including select strains among >30 distinct Arthrobacter isolates, will be examined for shared growth requirements with Deinococcus spp. to determine the inter-relationship between nutrient conditions (particularly Mn(II) availability), resistance to environmental stress (including variable water content), and metal reduction.
  4. Element: Biomolecular Sciences/Engineering. Characterizing the genetic basis of metal reduction and role of Mn in Arthrobacter aurescens (TC1) and novel Arthrobacter isolates from the Hanford Site. Like Deinococcus spp., Arthrobacter spp. are exceptionally robust, resistant to radiation and generally well-adapted to life in soil and vadose sediments. Isolates whose closest match was a member of the genus Arthrobacter were the most common for cultures originating from contaminated subsurface vadose sediments collected beneath tank SX-108 on the Hanford Site. Arthrobacter spp. have also recently been reported to efficiently resist and reduce high concentrations of hexavalent chromium. The whole genome sequence of Arthrobacter aurescens (TC1) is currently being acquired at The Institute for Genomic Research (TIGR) under an NSF grant led by Dr. Wackett. Comparative physiologic and genomic analyses of Deinococcus and Arthrobacter could help identify and delineate pathways that contribute to metal reduction in these bacteria, and the cellular processes that contribute to the remarkable environmental robustness of these organisms. Significantly, both groups appear to have metabolisms that are highly dependent on Mn(II).