Geobacter Sulfurreducens TIGR Complete

eobacter Project www.geobacter.org

Genomics-GTL Addressing DOE Environmental Science Needs in:

• In situ characterization of contaminated environments

• Understanding contaminant fate and transport

• Development of strategies for in situ control or remediation of contaminated sites Tools From Genomes-GTL Applicable to DOE Environmental Restoration Needs • In situ characterization of contaminated environments Molecular (mRNA) analysis of the in situ metabolic state of the microbial community via whole-genome analysis to reveal: -environmental stresses -nutrient requirements

• Understanding contaminant fate and transport Molecular (mRNA) analysis of in situ rates of metal reduction from levels of expression of key respiratory gene

• Development of strategies for in situ control or remediation of contaminated sites Prediction of fate of contaminants under natural attenuation or engineered bioremediation options by coupling in silico microbial models with the appropriate hydrological and geochemical models eobacter Project www.geobacter.org Principle Investigators Derek Lovley, UMASS, ecology, physiology, and biochemistry of Geobacteraceae Maddalena Coppi, UMASS, genetics of Geobacteraceae Stacy Ciufo, UMASS, bioinformatics, environmental genomics Barbara Methe, TIGR, bioinformatics, DNA microarray gene expression Pablo Pomposiello, UMASS, analysis of gene expression in response to stress Steve Sandler, UMASS, microbial genetics Cinthia Nunez, UMASS, microbial genetics, regulation of gene expression Daniel Bond, UMASS, anaerobic microbial physiology, electron transfer to electrodes Susan Childers, UMASS, microbial physiology, metabolic responses in Geobacter Carol Giometti, Argonne National Laboratory, proteomics Julia Krushkal, University of Tennessee, bioinformatics Christophe Shilling and Bernard Palsson, Genomatica, in silico modeling Analysis of the Genetic Potential and Gene Expression of Microbial Communities Involved in the In Situ Bioremediation of Uranium and Harvesting Electrical Energy from Organic Matter

The primary goal of this research is to develop conceptual and computational models that can describe the functioning of complex microbial communities involved in microbial processes of interest to the Department of Energy. Microbial Communities to be Investigated 1. Microbial community associated with the in situ bioremediation of uranium-contaminated groundwater. 2. Microbial community that is capable of harvesting energy from waste organic matter in the form of electricity.

DOE Needs Addressed 1. Remediation of metals and radionuclides at DOE sites 2. Development of cleaner forms of energy 3. Biomass conversion to energy Analysis of Microbial Communities Will Focus Exclusively on the Geobacter Component in the First Three Years

Rationale:

1. Geobacters account for ca. 50-90% of the total microbial community in the environments of interest.

2. Geobacters are the primary organisms carrying out the processes of interest in these environments.

3. Environmental genomics studies enhanced by parallel pure culture studies.

4. A meaningful evaluation of the other highly diverse components of the microbial communities in the environments of interest is not currently feasible. Microbial Bioremediation of Uranium

Acetate Carbon Dioxide Geo e bacter

U(VI) U(IV)

Uranium Contamination Removal Documented: Groundwaters from DOE Hanford Site

Surface water from DOI site

Washings from DOD contaminated soil

Lovley, D.R., E. J. P. Phillips, Y. A. Gorby, and E. R. Landa. 1991. Microbial reduction of uranium. Nature 350:413-416 InIn situsitu UraniumUranium BioremediationBioremediation StrategyStrategy Acetate Injection Geobacter species comprise as much as 85% of the microbial community in the subsurface during the most active phase of in situ uranium bioremediation.

Zone of U(VI) Removal

Threatened U(VI) U(VI) Down-Gradient Water Resource U(VI) U(VI)

2 CO2 Acetate

Geobacter Groundwater Flow

Fe(II) Fe(III)

U(IV) U(VI)

Anderson et al. 2003. Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer Appl. Environ. Microbiol. 69:5584-5891. Geobacter Can Use Electrodes as an Electron Acceptor Harvesting Power From Aquatic Sediments and Other Sources of Waste Organics

Cathode Reaction: + - 2O2 +8H + 8e Î 4H2O GeoBattery 4H2O 2O2

cathode water 8e- R 8H+ load sediment 1 - 10 cm

anode

Anode Reaction: e + - Geobacter species C2H4O2 + 2H2O Î 2 CO2 +8H + 8e Comprise ca. 50% of the Microbial Community Fermentation Sediment Acetate On the Anode 2 CO2 Organic Matter

Bond, Holmes, Tender, and Lovley. 2002. Science 295:483-485 Pure Culture Geobatteries Traditional Microbial Fuel Cell GeoBattery Organic Incomplete Organic Carbon Electron Oxidation Electron Dioxide Donor Products Donor

Direct Electron Oxidized Reduced Transfer to Electrode Electron Electron Shuttle Shuttle

ES H O H O ES 2 2

O2 O2

Anode Cathode Anode Cathode Comparison of Geo Batteries with Previous Microbial Fuel Cells Previous Microbial Fuel Cells GeoBattery

Oxidation of Incomplete Complete to Organic Fuel Carbon Dioxide

Requirement for Toxic Electron Shuttles Yes No to Function

Recovery of Electrons 1-50% 80-95% As Electricity

Long-Term Stability Poor Excellent

Ability to Function in No Yes “Open” Environments Potential Applications of GeoBatteries • Powering Monitoring Devices in Remote Locations

• Powering Electronic Devices from Renewable Energy Sources

• “Gastrobots”-robots fueled from food or organic waste

• Decentralized domestic power source

• Novel sensing devices

• Conversion of waste organic matter to electricity instead of methane

• Conversion of renewable biomass to electricity instead of ethanol

• Bioremediation of contaminated environments

• Powering automobiles Application of Environmental Genomics and Systems Biology to Uranium Bioremediation and Harvesting Electricity from Waste Organic Matter

Environmental Novel Culturing Genomic DNA of Strategies to Isolate Previously “As-Yet-Uncultured” Environmentally Relevant Cultured Geobacters Geobacters Geobacters

Genome Sequencing Functional Genomics Geobacter Genetic Potential Elucidation of Regulatory Systems Analysis of Gene Expression Physiological, Biochemical Studies Analysis of Gene Expression in Relevant Environments with Environmental Genome Arrays In Silico Model of Cell Function and Proteomics

In Silico and Conceptual Models for Optimizing Uranium Bioremediation and Electrical Energy Harvesting Application of Environmental Genomics and Systems Biology to Uranium Bioremediation and Harvesting Electricity from Waste Organic Matter

Environmental Novel Culturing Genomic DNA of Strategies to Isolate Previously “As-Yet-Uncultured” Environmentally Relevant Cultured Geobacters Geobacters Geobacters

In Silico and Conceptual Models for Optimizing Uranium Bioremediation and Electrical Energy Harvesting Status of Geobacteraceae Sequencing Projects

Organism Sequencing Status Group

Geobacter sulfurreducens TIGR Complete

Geobacter metallireducens JGI/UMASS Nearly Complete

Desulfuromonas acetoxidans JGI/UMASS 10 gaps

Pelobacter carbinolicus JGI/UMASS Draft

Pelobacter propionicus JGI/UMASS Draft

Geobacter uranibioremediacens JGI/UMASS Underway Status of Geobacteraceae Sequencing Projects

Organism Sequencing Status Group

Geobacter sulfurreducens TIGR Complete

Geobacter metallireducens JGI/UMASS Nearly Complete

Desulfuromonas acetoxidans JGI/UMASS 10 gaps

Pelobacter carbinolicus JGI/UMASS Draft

Pelobacter propionicus JGI/UMASS Draft

Geobacter uranibioremediacens JGI/UMASS Underway DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR • Demonstration that Geobacter species can grow with oxygen as the terminal electron acceptor

• Elucidation of novel mechanism for Geobacter species to find and access Fe(III) oxides

• Elucidation of genes encoding for key respiratory proteins

• Elucidation of systems regulating expression key respiratory genes

• Elucidation of systems regulating: growth under slow, environmentally relevant conditions response to environmental stress response to nutrient limitation

• Elucidation of novel central metabolism genes DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR (continued)

• Development of an in silico model that can:

-- Predict the response of Geobacter to different environmental conditions including strategies for manipulating the environment to promote bioremediation

-- Aid in elucidating the likely outcome of genetically engineering novel metabolic capabilities in Geobacter

• Discovery of significant similarities in genomes of as-yet-uncultured Geobacter species and pure cultures of Geobacter species DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR • Demonstration that Geobacter species can grow with oxygen as the terminal electron acceptor

Provides explanation for the reservoir of Geobacter species in aerobic aquifers that can so rapidly respond to introduction of acetate and immediately start removing uranium from contaminated groundwater. Genome-based Model for the Reduction of Oxygen and the Detoxification of Reactive Oxygen Species by G. sulfurreducens

Outer membrane superoxide cyto c NiFe reductase peroxidase Hydrogenase(s) O • - + 2H+ 2 H O 2 H2O2 2 H2 + O2 2 H2O + 2H+ Cyt C

Cyto c Inner membrane Cyto ? bd bc1 oxidase

Outer membrane superoxide cyto c NiFe reductase peroxidase Hydrogenase(s) O • - + 2H+ 2 H O 2 H2O2 2 H2 + O2 2 H2O + 2H+ Cyt C

Cyto c Inner membrane Cyto ? bd bc1 oxidase

2 O 2 H O 2 2 H2O ATP 2 +4 H+ superoxide rubrerythrin(s) + • - dismutase H2O2 + 2H 2 H2O O2 + H2 O2 + H2O2 thioredoxin peroxidase(s) NADH + H2O2 + 2H 2 H2O oxidase(s) catalase + + O2 + NADH +H NAD + H + H2O2 2 H2O2 O2 + H2O Growth of G. sulfurreducens can grow with oxygen as the sole terminal electron acceptor Growth of Geobacter on oxygen provides an explanation for how Acetate Geobacter survives in low numbers in aerobic Growth subsurface environments and then rapidly responds to the development of anaerobic conditions when uranium bioremediation is initiated. Lin, Coppi, and Lovley. 2004. Geobacter sulfurreducens can grow with oxygen as a terminal electron acceptor. Appl. Environ. Microbiol. 70: (in press). Knocking out the cytochrome oxidase genes inhibits growth of G. sulfurreducens on oxygen • Cytochrome oxidase is comprised of four genes, ORFs 374, 376, 378, and 380. Wild Type Growth • Mutant is a deletion of 374, 376 and replaced with antibiotic Mutant Growth resistance cassette.

• Mutant does not grow with

O2 but still can consume O2.

• Implications: -Terminal oxidase is responsible for growth

with low % O2.

- Inactivation of terminal oxidase does not affect the activity of oxidative stress enzymes. DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR • Elucidation of novel mechanism for Geobacter species to find and access Fe(III) oxides

Solves the mystery of how Geobacter species, which were thought to be non-motile, can efficiently access Fe(III) oxides via chemotaxis and thus compete for Fe(III) oxides even though Geobacter species require direct contact with Fe(III) oxides in order to reduce them. Fe(II)

Fe (III) Geobacter Specifically Expresses Oxide Flagella when only Fe(III) Oxide is Available as an Electron Acceptor Geobacter follows Fe(II) Gradient to Locate Fe(III) Oxides

Fe (III) Fe (III) Oxide Oxide Fe (III) Oxide

Geobacter May use Flagella to Make Initial Contact with Fe(III) Geobacter uses Pili to “Twitch” Along Sediment Surface and Contact Fe(III)

Childers S.E., S. Ciufo, and D. R. Lovley. 2002. Geobacter metallireducens access Fe(III) oxide by chemotaxis. Nature 416:767-769 DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR

• Elucidation of genes encoding for key respiratory proteins

Provides molecular targets for estimating rates of metal reduction in the subsurface. Electron Transfer to Extracellular Electron Acceptors Such as Metals and Electrodes is Fundamentally Different than the Reduction of Commonly Considered Soluble Electron Acceptors Fe(III) O2, NO3, SO4 Fe(II)

???

Reduction at inner membrane or in the cytoplasm 160 r2= 0.70 p< 0.01 GS 140

AF 120

PA 100 Geobacter sulfurreducens has a unusually high percentage of genes devoted to electron transport

80 many of which encode for c-type cytochromes

60 AA

0 0 1000 2000 3000 4000 5000 6000 Total # of ORFs in each Organism ModelModel forfor ElectronElectron TransferTransfer toto NADH Fe(III)Fe(III) inin GeobacterGeobacter DH

NAD Fe(III) MQ ox OmpB MQ Fe(III) red omcB OmcD M OmpA acA ,D,E A,C OmcE Ppp Fe(II) OmcBOmcB butbut notnot OmcCOmcC isis RequiredRequired forfor Fe(III)ReductionFe(III)Reduction

60 ∆omcC:: kan 50

Fe(II) mM 20 Wild type 10 ∆omcB::cam 0 0 20 40 60 80 100 120 Hours

Leang, C., M. V. Coppi, and D. R. Lovley. 2003. OmcB, a c-Type polyheme cytochrome, involved in Fe(III) reduction in Geobacter sulfurreducens. J. Bacteriol. 185:2096-2013. Direct Correlation Between levels of omcB mRNA and Rates of Fe(III) Reduction in Acetate-Limited Chemostats of Geobacter sulfurreducens ) 5.00E+05

4.00E+05

3.00E+05 RNA total g µ

2 transcripts 2.00E+05 R = 0.9355

1.00E+05 omcB

0.00E+00 (copy numbers/ 0.1 0.15 0.2 0.25 0.3 0.35

Fe(III) reduction rate (mmol electrons/mg protein/h) DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR

• Elucidation of systems regulating expression key respiratory genes

This makes it possible to predict under which environmental conditions respiratory genes necessary for metals bioremediation will be expressed. ENVIRONMENTAL STIMULI RESPONSE

Signals Differential gene expression REGULATORY CASCADES TO CONTROL TRANSCRIPTION

SIGMA FACTORS TWO COMPONENT OTHER GLOBAL SYSTEMS REGULATORS RpoS RpoE Histidine kinases RelA Fur Response regulators

ELECTRON TRANSFER Defining the RpoS regulon in G. sulfurreducens RNA pol RpoS Genetics: C. Nunez B. Methe TARGET GENE MICROARRAYS -35 -10 EXPRESION

PROTEOMICS: C. Giometti

Wildtype RpoS mutant

• Stationary phase may more closely represent physiological state in subsurface environments or on electrodes • Knocking out rpoS affects the expression of at least 100 other genes • Genes regulated include cytochrome genes required for Fe(III) reduction • First definition of role of rpoS in δ-proteobacteria Defining the RpoE regulon in G. sulfurreducens RNA pol RpoE An rpoE mutation affects the TARGET GENE expression of at least 200 other genes -35 -10 EXPRESION MICROARRAYS • RpoE REGULON: 1. Cytochrome genes (7) and cytochrome biogenesis genes involved in Fe(III) reduction 2. Oxidative stress regulon different from RpoS 3. Biofilm metabolism and development

WT RpoE-

50 µm

4. Biofilm electron transfer via H2 Rel A Plays an Important Role in Regulating Growth and Metabolism in Geobacter sulfurreducens under Environmentally Relevant Conditions Starvation Phenotype of relA Mutant • Increase growth under nutrient limitation GTP RelA • Decrease growth in presence of oxygen

ppGpp • Upregulation of genes involved in: protein biosynthesis cell division transport • Downregulation of genes for: RNA pol ppGpp stress response Target gene signal transduction insoluble Fe(III) reduction

Slower Growth (protein synthesis, nutrient transport) Increased resistance to Oxidative Stress Resistance Increased production of cytochromes for Fe(III) Reduction Microarray Results Comparing Wild Type to the fur Mutant

Regulators Up regulation of 9 regulatory genes was found, including dtx, another iron regulated repressor. Metabolism Up regulation of 15 genes involved in metabolism including HydB, the hydrogenase responsible for hydrogen dependant growth. Metal Uptake Up regulation of 16 possible metal uptake genes was found, including FeoB,a ferrous iron cytoplasmic membrane transporter. Cytochromes Up regulation of 7 cytochrome genes was found, including OmcB and OmcD.

Unknown Proteins Up regulation of 32 genes with an unknown function. Knocking out a Histidine Kinase Sensor Inhibits Cytochrome Production

WT 941 Mutant

1-D and 2-D SDS-PAGE stained for c-type cytochromes (heme) Fe(III)-Specific Regulation of Fumarate Respiration

frdCAB mRNA levels 14 Fe pulse control 8

12 succinate 6 Fe(II)

Fe(II) mM 4 Succinate (mM)

0 0 0 5 10 15 20 25 30 Time (h) Fe(III) pulse Abraham Esteve-Núñez, Cinthia Núñez and Derek R. Lovley. 2004. J. Bacteriol. (in press). DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR

• Elucidation of systems regulating: growth under slow, environmentally relevant conditions response to environmental stress response to nutrient limitation

Provides information necessary to interpret the in situ metabolic state of Geobacter species in the subsurface. DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR

• Elucidation of novel central metabolism genes

For example, the novel, eucaryotic-like citrate synthase provides a unique molecular marker for tracking Geobacter species and their activity in the subsurface. DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR (continued)

• Development of an in silico model that can:

-- Predict the response of Geobacter to different environmental conditions including strategies for manipulating the environment to promote bioremediation

-- Aid in elucidating the likely outcome of genetically engineering novel metabolic capabilities in Geobacter ContributionsContributions ofof IterativeIterative InIn SilicoSilico ModelModel BuildingBuilding toto UnderstandingUnderstanding ofof thethe EnvironmentalEnvironmental ResponsesResponses ofof GeobacterGeobacter

in silico-based hypothesis Computational Biochemical Experiment Experiment Prediction

Genome Sequence in silico In vitro/in vivo Information Cellular Models characteristics

Refinement Revised Genome Inferred Fitness & Annotations Capabilities

Added Network Function Genome-scale model of G. sulfurreducens

Total Number of Genes: 3532 Included Genes: 583 (17 %) Percentage of the annotated genome: (29%) Total Number of Reactions: 520 Gene/(Non-gene) associated: 466 (54) Number of Proteins: 431 Number of Metabolites: 537 Given a limited number of constraints, growth rate, yield, and flux through metabolic network can be quantitatively predicted

Predicted growth rate vs. observed Flux (example) Phylogenetically Distinct Fe(III) Reducers Have Different Mechanisms for Fe(III) Reduction Fe(II) Geobacter has to directly contact Fe(III) oxide in order to reduce it

Fe (III) Nevin, K. P., and D. R. Lovley. 2000. Oxide Fe(III) Appl. Environ. Microbiol. 66:2248-2251 Childers S.E., S. Ciufo, and D. R. Lovley. 2002. Nature 416:767-769 Geothrix releases electron shuttles and chelators which alleviate the need for direct microbe-Fe(III) oxide contact

C < Release of Fe(II) Shuttle Microbially Fe(II) C< Fe(III) Fe (III) Produced Oxide Reduced ) Shuttle Fe (III) Oxide

Nevin, K. P., and D. R. Lovley. 2002. Appl. Environ. Microbiol. 68:2294-2299 Analysis of Metabolic Cost to Release a Quinone-Based Electron Shuttle Energetics of Menaquinone Secretion (10 mmol/gdw hr Acetate Uptake) Simulations carried out for varying • Quinone secretion rates 0.9-1 0.8-0.9 • Different Sized Molecules 0.7-0.8 1 0.6-0.7 0.9 0.5-0.6 0.8 0.4-0.5 Significant growth rate 0.3-0.4 0.7 0.2-0.3 reduction due to both ATP 0.6 0.1-0.2 requirements and carbon 0.5 0-0.1 0.4 requirements for shuttle 0 0.02 0.3 Normalized synthesis Growth Rate 0.04 0.2 Menaquinone 0.06 0.1 Secretion Rate 0.08 0 (mmol/gdw hr) 0.1 Provides likely explanation for MQN9

MQN8 the predominance Geobacter MQN7 MQN6 MQN5 MQN4

MQN3 Number of Units over Geothrix in subsurface environments DiscoveriesDiscoveries fromfrom GTLGTL withwith DirectDirect andand ImmediateImmediate ApplicationApplication toto NABIRNABIR (continued)

• Discovery of significant similarities in genomes of as-yet-uncultured Geobacter species and pure cultures of Geobacter species

Suggests that models based on intensively studied pure cultures may have applicability to predicting the activity of as-yet-uncultured Geobacter species that predominate in uranium-contaminated subsurface environments. Geobacter uranibioremediacens: Isolate from Rifle, Colorado Field Site

16S rDNA sequence identical with predominant Geobacter sequence in groundwater during uranium bioremediation

Direct Isolation on Solidified Phase Contrast Micrograph of Cells Medium with Aquifer Clay Amongst Sediment Clay Fraction Fraction Serving as Fe(III) Source In Ground-Water Amended Medium BAC clone from as-yet- gyrB gyrA gspA 16S trna 23S trna 5S uncultured Geobacter from subsurface GDEF protein Stress response protein sediments contains ATP binding permease Metal binding protein ABC transporter omcB, a gene for an adenosyltransferase aminotransferase periplasmic protein outer-membrane cytochrome required for Fe(III) reduction orf1 orf2 omcB orf3 orf4 omcC

oxidoreductase transporter in G. sulfurreducens transporter inner membrane protein in the same gene organization as seen in lexA dinP G. sulfurreducens dnaJ dnaN recF dnaK RNA ligase

tbB tolQ hrcA grpE

hydrolase histidine kinase response regulator

protophoryin oxidase marR Map of BAC 109

membrane efflux efflux efflux ompB ompB lipoprotein protein protein protein protein

efflux sensory box protein tetR histidine kinase LuxR

dehydropantoate reductase

response hlyD regulator

drug drug acrB resistance resistance tetR

moeA

fibronectin sigma-54 dependent DNA-binding response regulator alpha amylase alpha amylase

thiamine- selenide dikinase phosphatepyrophosphorylase

phosphoribosylaminoimidazole- carboxamideformyltransferase resB

ppcA

glycosyltransferase lipopolysaccharide transporter

glycosyltransferase

signal recognition particle protein tRNA(guanine-N1)-mehtyltransferase

ribonuclease hydrolase tetrapyrrole methylase General Secretion Pathway

hypothetical gspL gspK gspJ BAC 83 % Similarity 67% 56% 63.6% 52.4% gspI gspH gspG gspF gspE gspD

67.2%73% 62.8% 65.8% 66.4% 63.8%

hypothetical gspL gspK gspJ G. sulfurreducens

gspI gspH gspG gspF gspE gspD Flagella

flgB flgC fliE fliF fliG fliH fliJ BAC 84 % Similarity 61.9% 65% 63% 71.9% 67% 66.2% 66% flgD flgE fliL fliM (end of Bac) 52.1% 66.3% 63.3% 63.2%

flgB flgC fliE fliF fliG fliH fliJ G. sulfurreducens

flgD flgE fliL fliM fliN fliP fliQ fliR flhB n

i 62.2%

p p 56.5%

Pilin

p p

60.5%

p p 62.6% % Similarity BAC 96 G. sulfurreducens Cytochrome Oxidase

subunit subunit subunit subunit 1 3 4 2 BAC 82

% Similarity 64.5% 60% 62.5% 66.7%

subunit subunit subunit subunit G. sulfurreducens 1 3 4 2 Hydrogenase A

Maturation Large Small Competence protein subunit subunit F BAC 81

% Similarity 56% 66.2% 58.2% 64%

Maturation Large Small Competence protein subunit subunit F G. sulfurreducens Hydrogenase B

Small FeS Membrane Large subunit subunit protein subunit BAC 88

% Similarity 63.8% 68.5% 61% 63.7%

Small FeS Membrane Large G. sulfurreducens subunit subunit protein subunit D

6 f Formate Dehydrogenase BAC 147 G. sulfurreducens Nitrogen Fixation Genes

nifK nifD nifH BAC 74

% Similarity 62% 63% 61.9%

nifK nifD nifH G. sulfurreducens Application of Environmental Genomics and Systems Biology to Uranium Bioremediation and Harvesting Electricity from Waste Organic Matter

Environmental Novel Culturing Genomic DNA of Strategies to Isolate Previously “As-Yet-Uncultured” Environmentally Relevant Cultured Geobacters Geobacters Geobacters

In Silico and Conceptual Models for Optimizing Uranium Bioremediation and Electrical Energy Harvesting nifD and recA expression in Acetate-amended and Control Subsurface Sediments

before and after adding 100 µM NH4Cl 7 10 acetate (nif) control (nif) 6 acetate (rec) 10 control (rec)

5 10

4 g of total RNA 10 µ

1000

100 mRNA expression per 10

Day 0 Day 1 Day 2 Geobacter Genes Upregulated During Growth on Electrodes heat shock protein, Hsp20 family dnaJ domain protein heat shock protein, Hsp20 family heat shock protein, Hsp20 family heat shock protein, Hsp20 family cytochrome c family protein hypothetical protein NOL1/NOP2/sun family protein conserved domain protein C4-dicarboxylate transporter, anaerobic cytochrome c family protein, putative cytochrome c family protein ClpB protein NHL repeat domain protein metal ion efflux outer memb. prot. family, put. hypothetical protein hypothetical protein ABC transporter, permease protein transcriptional regulator, MerR family hypothetical protein Geobacter Genes Upregulated During Growth on Electrodes heat shock protein, Hsp20 family dnaJ domain protein heat shock protein, Hsp20 family heat shock protein, Hsp20 family heat shock protein, Hsp20 family cytochrome c family protein OmcD hypothetical protein NOL1/NOP2/sun family protein conserved domain protein C4-dicarboxylate transporter, anaerobic cytochrome c family protein, putative cytochrome c family protein ClpB protein NHL repeat domain protein metal ion efflux outer memb. prot. family, put. hypothetical protein hypothetical protein ABC transporter, permease protein transcriptional regulator, MerR family hypothetical protein Effect of Deletion Mutation in omcD on

1 Current Production

0.8 Wild type

0.6 OmcD Mutant-Complemented

0.4

Current (mA) Current OmcD Mutant 0.2

0 0 50 100 150 200 250 Hours Support of NABIR by GTL in the Future • Use of molecular techniques to assess in situ rates of metal reduction.

• Whole-genome analysis of in situ gene expression to determine the in situ metabolic state of microorganisms during uranium bioremediation which will help direct implementation of bioremediation strategies.

• Coupling in silico microbial models with geochemical and hydrological models to accurately predict the rate and extent of bioremediation in diverse environments under various bioremediation strategies.