Applied Environmental

Microbiology Core

Terry C. Hazen (LBNL, Core Team Leader)Matthew Fields (ORNL)Hoi-Ying Holman (LBNL)

Martin Keller (Diversa Inc.)David Stahl (University of Washington)

Dorothea Thompson (ORNL)Judy Wall (University of Missouri)

Jizhong Zhou (ORNL)

U Washington

U Missouri

(Consultant)

AEMC Mission

• AEMC is the source of environmental data and samples that determine the stressors that will be studied, provides the environments for growing the organisms to be tested, simulates stressed environments, and verifies the conceptual models to determine how these stress regulatory pathways control the biogeochemistry of contaminated sites

Main Goals

• Develop criteria for monitoring the integrity (health) and altering the trajectory of an environmental biological system (process control).

• More complete understanding of the diversity and environmental context of stress response.

Specific Aims

1. Survey and map DOE sites contaminated by metals and radionuclides using chemical and molecular/ microbiological parameters to determine major microbial populations and potential stressors for Desulfovibrio vulgaris, Geobacter metallireducens, and Shewanella oneidensis MR1.

2. Determine the rank priority of these stressors in terms of their ability to affect metal/radionuclide bioreduction by either direct or indirect processes, and to establish the normal active range of the stressor in metal/radionuclide contaminated environments.

3. Determine the incidence and activity of the three target bacteria, and closely related relatives, in the test metal/radionuclide contaminated environments and collect isolates for analysis by the Functional Genomics Core for comparison using 16S RNA profiling.

Specific Aims, cont.

4. Create defensible environmental simulators that can replicate key features of field site chemical and biological structure to mimic stress conditions for single populations and later for microbial communities (chemostats to soil columns from 10 µm to 1 m size systems).

5. Provide large quantities of cells in various stress states for the Functional Genomics Core’s physiological monitoring facility, and molecular interaction studies.

6. Provide environmental simulators for testing stressor effects on mutants, large insert clones, expression analysis, etc., for elucidating critical parts of the stress regulation pathway.

7. Develop testable conceptual models of stress regulatory pathways based on results of the Computational Core that could predict natural attenuation and suggest biostimulatory strategies for immobilization of metals and radionuclides at DOE contaminated sites.

Specific Aims, cont.

8. Test conceptual models of stress regulatory pathways and effects on contaminate site biogeochemistry using competent soil columns with different levels of complexity over the active range of the stressors

9. Validate conceptual models using field tests at contaminated sites that utilize specific functional gene arrays developed from the stress regulatory pathways.

10. Alter field conditions or test along gradients to verify stress regulatory model efficiency for predicting natural attenuation or suggesting biostimulatory strategies for immobilization of metals and radionuclides.

Research Elements

1. chemical and molecular/microbiological mapping of field sites to identify major microbial populations and potential system stressors

2. development of reactor systems that replicate key features of field site chemical and biological structure

3. testing of different stressors using appropriate controlled reactor systems in combination with a general measure of physiological stress (rRNA synthesis)

4. development of simplified reactor systems containing the target organism, mutant variants, and native populations that are closely related to the target organism

5. field validation of conceptual models using chemical and molecular/microbiological probes and assays based on the stress regulatory pathways developed during the overall project.

Chemical and Molecular Microbiological Mapping of Field Sites

• NABIR Field Research Center at Oak Ridge

• Pit 7 Complex at Site 300 of LLNL• General sediment and soil sampling• Chemical analyses of field sites• TRFLP and Clonal Population Mapping

Reactor System Development

• Fixed bed reactors• Fluidized bed reactors• Soil columns• Rationale:

– Analogous to saturated soil/sediment systems in which the microbiota colonize surfaces as biofilms

– Growth as biofilms is the more common state for microorganisms in the environment

– Fluidized bed reactor systems provide for experimental control, replication, high biomass, and ease of sampling

– Biofilm-associated populations are more resistant to stress and are the more relevant state for most environmental populations

– Biofilm-associated populations are not washed out (of the reactor system) following imposition of stress, providing for greater range of stress-response experimentation

– Past reactor studies have shown that Desulfovibrio spp. and Geobacter-like populations will colonize and be retained within these systems

Reactors cont.

• Chemostat Studies• Cultural isolation of organisms for

comparative “Benchmarks”• Collection of environmental samples for

reactor inoculation• Comparability of reactor and field site

community structure

Environmental Simulator Stress Analyses

• Growth and recovery of stressed single populations under simple conditions

• Identification and recovery of stress-responsive populations from complex communities

• Radiomicroarray analysis• Recover of stress responsive populations via

flow cytometry

Simulator Analyses, cont.

• Flow cytometric sorting of FISH-labeled populations

• Expresssion analysis of RNA extraction from sediment columns and reactor systems

• Synchrotron FTIR direct analysis of stress changes in living cells

• Direct stress and community comparison with PLFA and metabolite analyses

Validation of Conceptual Models with Simulators and Field Tests

• Subjecting various reactor systems to stress and recovery scenarios as predicted by models

• Use of new molecular probes to verify responses and pathways

• Push-pull tests along defined stressor gradients in field tests

• Induce stressor change with amendments and verify predicted response

Experimental Core Facilities

• Environmental Molecular Microbiology Facility (ORNL, LBNL, U. Wash., Diversa Inc.)

• Environmental Simulation and Culture Facility (LBNL, U. Wash., Diversa Inc.)

AEMC MilestonesOakridge National Laboratory (Note: Expression profiling will be done in part with

environmental simulators, these milestones are shared with the Functional Genomics Core)

Year 1 (July - September, 2002): Begin survey and mapping of ORNL sites for stressors and target organisms. Obtain isolates from site for culturing. Initial expression profiling experiments with S. oneidensis

Year 2 (October, 2002 – September 2003): Continue survey and mapping of ORNL site. Expression profiling experiments with S. oneidensis; Initial expression profiling experiments with D.vulgaris and G. metallireducens

Year 3 (October, 2003 – September 2004): Expression profiling experiments with S. oneidensis; Expression profiling experiments with regulatory mutants of S. oneidensis; Expression profiling experiments with D.vulgaris and G. metallireducens

Year 4 (October, 2004 – September 2005): Expression profiling experiments with regulatory mutants of S. oneidensis; Expression profiling experiments with D.vulgaris and G. metallireducens

Year 5 (October, 2005 – September 2006): Expression profiling experiments with regulatory mutants of S. oneidensis; Expression profiling experiments with D.vulgaris and G. metallireducens; Expression profiling experiments with regulatory mutants of D.vulgaris and G. metallireducens. Begin testing of conceptual models in the field at ORNL using new probes and assays. Provide field and lab data to Computational Core.

Year 6 (October, 2006 – September 2007): Expression profiling experiments with D.vulgaris and G. metallireducens; Expression profiling experiments with regulatory mutants of D.vulgaris and G. metallireducens. Begin testing of conceptual models in the field at ORNL using new probes and assays. Provide field and lab data to Computational Core. Survey and map other contaminated DOE sites for verification of general applicability of conceptual models using new probes and assays.

AEMC Milestones, cont.

University of WashingtonYear 1 (July - September, 2002): Establish high-speed sorting

capability

Year 2 (October, 2002 – September 2003): Characterize microbiological and chemical structure of field sites; Recovery of specific population by high-speed sorting

Year 3 (October, 2003 – September 2004): Development of DNA microarray probe set; Bioreactor set-up and operation

Year 4 (October, 2004 – September 2005): Identify stress-responsive reactor populations

Year 5 (October, 2005 – September 2006): Recovery of specific populations by high-speed sorting; Associate sorted populations with specific stress-response genes;

Year 6 (October, 2006 – September 2007): Verify and validate conceptual models at field sites using new Microarrays and sorting techniques.

AEMC Milestones, cont.University of Missouri at Columbia Note: Environmental simulator, chemostat and

field testing will also involve Dr. Wall, so her milestones are also in the Functional Genomics Core where the bulk of her tasks are associated.

Year 1 (July - September, 2002): Develop conjugation system for D. vulgaris. Begin development of DNA microarrays for D. vulgaris.

Year 2 (October, 2002 – September 2003): Disrupt the restriction-modification system in D. vulgaris. Continued development of DNA microarrays for D. vulgaris.

Year 3 (October, 2003 – September 2004): Develop a system that will allow transformation of D. vulgaris with dsDNA and double recombination. DNA microarray analysis of wild-type D. vulgaris subjected to various stresses.

Year 4 (October, 2004 – September 2005): Develop a transposon mutagenesis system for D. vulgaris. Identify and clone regulated promoters for D. vulgaris. Further DNA microarray analysis of wild-type D. vulgaris subjected to various stresses.

Year 5 (October, 2005 – September 2006): Make mutations in various stress-response related genes in D. vulgaris. DNA microarray analysis of small molecule-inhibited D. vulgaris subjected to various stresses.

Year 6 (October, 2006 – September 2007): DNA microarray analysis of mutant D. vulgaris subjected to various stresses.

AEMC Milestones, cont.Lawrence Berkeley National LaboratoryYear 1 (July - September, 2002): Begin survey and mapping of ORNL sites for stressors and target organisms. Obtain isolates from site

for culturing. Set up environmental simulator facility with PLFA and scanning laser confocal microscopy. Do preliminary studies to determine protocols for target bacteria for SFTIR spectra and unique signatures. Begin development of environmental chambers. Provide field data to Computational Core.

Year 2 (October, 2002 – September 2003): Continue survey and mapping of ORNL site and begin survey and mapping at LLNL site. Obtain isolates from LLNL site for culturing. Establish rank priority of stressors at the ORNL site. Continue development of environmental chambers. Analyze SFTIR and PLFA signatures for stressed and unstressed bacteria. Initiate stressor studies in chemostats and soil columns for expression analysis, PLFA, SFTIR, etc. Begin cell production for Functional Genomics Core. Provide field and lab data to Computational Core.

Year 3 (October, 2003 – September 2004): Continue survey and mapping of LLNL for stressors and target organisms. Continue development of environmental chambers. Continue stressor studies in chemostats and soil columns for expression analysis, PLFA, SFTIR, etc. Analyze SFTIR and PLFA signatures for stressed and unstressed bacteria. Continue cell production for Functional Genomics Core. Provide field and lab data to Computational Core.

Year 4 (October, 2004 – September 2005): Continue stressor studies in chemostats and soil columns for expression analysis, PLFA, SFTIR, etc. Continue cell production for Functional Genomics Core. Determine incidence of closely related relatives to target bacteria in chemostats, soil columns and field samples. Analyze SFTIR and PLFA signatures for stressed and unstressed bacteria. Continue cell production for Functional Genomics Core. Provide lab data to Computational Core.

Year 5 (October, 2005 – September 2006): Continue stressor studies in chemostats and soil columns for expression analysis, PLFA, SFTIR, etc. Continue cell production for Functional Genomics Core. Determine incidence of closely related relatives to target bacteria in chemostats, soil columns and field samples. Analyze SFTIR and PLFA signatures for stressed and unstressed bacteria. Continue cell production for Functional Genomics Core. Begin testing of conceptual models in both the laboratory and field using new probes and assays. Provide field and lab data to Computational Core.

Year 6 (October, 2006 – September 2007): Continue stressor studies in chemostats and soil columns for expression analysis, etc. Continue cell production for Functional Genomics Core. Determine incidence of closely related relatives to target bacteria in chemostats, soil columns and field samples. Continue cell production for Functional Genomics Core. Continue testing of conceptual models in both the laboratory and field using new probes and assays. Provide field and lab data to Computational Core. Survey and map other contaminated DOE sites for verification of general applicability of conceptual models using new probes and assays.