eobacter Project · GeoBattery e Bond, Holmes, Tender, and Lovley. 2002. Science 295:483-485...

eobacter Projectwww.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 ofcontaminated sites

Tools From Genomes-GTL Applicable to DOE Environmental Restoration Needs

• In situ characterization of contaminated environmentsMolecular (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 transportMolecular (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 ofcontaminated 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 Projectwww.geobacter.org

Principle InvestigatorsDerek Lovley, UMASS, ecology, physiology, and biochemistry of GeobacteraceaeMaddalena Coppi, UMASS, genetics of GeobacteraceaeStacy Ciufo, UMASS, bioinformatics, environmental genomicsBarbara Methe, TIGR, bioinformatics, DNA microarray gene expressionPablo Pomposiello, UMASS, analysis of gene expression in response to stressSteve Sandler, UMASS, microbial geneticsCinthia Nunez, UMASS, microbial genetics, regulation of gene expressionDaniel Bond, UMASS, anaerobic microbial physiology, electron transfer to electrodesSusan Childers, UMASS, microbial physiology, metabolic responses in GeobacterCarol Giometti, Argonne National Laboratory, proteomicsJulia Krushkal, University of Tennessee, bioinformaticsChristophe 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 conceptualand computational models that can describe the functioning of complexmicrobial communities involved in microbial processes of interest tothe Department of Energy. Microbial Communities to be Investigated1. 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 Addressed1. Remediation of metals and radionuclides at DOE sites2. Development of cleaner forms of energy3. 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 communityin 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 notcurrently feasible.

Geo bacter

Acetate Carbon Dioxide

U(VI) U(IV)

e

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

Microbial Bioremediation of Uranium

Zone of U(VI) Removal

AcetateInjection

U(VI)

U(VI)U(VI)

Threatened Down-GradientWater Resource

Geobacter

U(VI)

Acetate2 CO2

Fe(III)Fe(II)

U(IV)

GroundwaterFlow

U(VI)

In situ In situ Uranium Bioremediation StrategyUranium Bioremediation Strategy

Geobacter species comprise as much as 85% of the microbial community in the subsurface during the most active phase of in situ uranium bioremediation.

Anderson et al. 2003. Stimulating the in situ activity of Geobacter species to remove uranium from thegroundwater of a uranium-contaminated aquifer Appl. Environ. Microbiol. 69:5584-5891.

Rload1 - 10 cm

Geobacter Can Use Electrodes as an Electron AcceptorHarvesting Power From Aquatic Sediments and Other Sources of Waste Organics

anode

8e-

Acetate2 CO2

4H2O

8H+

Anode Reaction:C2H4O2 + 2H2O 2 CO2 +8H+ + 8e-

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

water

sediment

cathode

2O2GeoBattery

e

Bond, Holmes, Tender, and Lovley. 2002. Science 295:483-485

Geobacter species Comprise ca. 50% of theMicrobial CommunityOn the Anode

SedimentOrganic Matter

Fermentation

Pure Culture Geobatteries

ES

ESO2

H2O

O2

H2O

IncompleteOxidationProducts

OrganicElectronDonor

Oxidized Electron Shuttle

ReducedElectronShuttle

OrganicElectron Donor

CarbonDioxide

Direct ElectronTransfer to Electrode

Traditional Microbial Fuel CellTraditional Microbial Fuel Cell GeoBatteryGeoBattery

Anode Cathode Anode Cathode

Previous Microbial Fuel Cells GeoBattery

Oxidation of Incomplete Complete to Organic Fuel Carbon Dioxide

Requirement for ToxicElectron Shuttles Yes Noto Function

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

Long-Term Stability Poor Excellent

Ability to Function in No Yes“Open” Environments

Comparison of Geo Batteries with Previous Microbial Fuel CellsComparison of Geo Batteries with Previous Microbial Fuel Cells

Potential Applications of Potential Applications of GeoBatteriesGeoBatteries• 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

EnvironmentalGenomic DNA of“As-Yet-Uncultured”Geobacters

Geobacter Genetic Potential

Novel CulturingStrategies to IsolateEnvironmentally RelevantGeobacters

Previously Cultured Geobacters

Analysis of Gene Expression in Relevant Environments with Environmental Genome Arraysand Proteomics

Functional GenomicsElucidation of Regulatory SystemsAnalysis of Gene ExpressionPhysiological, Biochemical Studies

In Silico Model of Cell Function

In Silico and Conceptual Models for OptimizingUranium Bioremediation and Electrical Energy Harvesting

Genome Sequencing

Application of Environmental Genomics and Systems Biology to UraApplication of Environmental Genomics and Systems Biology to Uranium nium Bioremediation and Harvesting Electricity from Waste Organic MatBioremediation and Harvesting Electricity from Waste Organic Matterter

Status of Geobacteraceae Sequencing Projects

Organism Sequencing StatusGroup

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

Discoveries from GTL with Direct and Immediate Discoveries from GTL with Direct and Immediate Application to NABIRApplication to NABIR

• Demonstration that Geobacter species can grow with oxygen as theterminal electron acceptor

• Elucidation of novel mechanism for Geobacter species to find andaccess 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 conditionsresponse to environmental stressresponse to nutrient limitation

• Elucidation of novel central metabolism genes


(continued)(continued)

• Development of an in silico model that can:

-- Predict the response of Geobacter to different environmentalconditions including strategies for manipulating the environment topromote bioremediation

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

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


• Demonstration that Geobacter species can grow with oxygen as theterminal 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.

Outer membrane

Genome-based Model for the Reduction of Oxygen and the Detoxification of Reactive Oxygen Species by G. sulfurreducens

O2• - + 2H+

superoxidereductase

H2O2

cyto cperoxidase

2 H2O 2 H2O

Inner membrane

Cyt C

2 H2O

2 O2+4 H+

2 H2O

Cyto coxidase

Cytobc1

H2 + O2+ 2H+

ATP

NiFe Hydrogenase(s)

bd

O2• - + H2

superoxidedismutase

O2 + NADH +H+ O2 + H2O

H2O2 + 2H+rubrerythrin(s)

O2 + H2O2

NAD + H+ + H2O2

H2O2 + 2H+NADHoxidase(s)2 H2O2

thioredoxinperoxidase(s)

2 H2Ocatalase

2 H2O

?

Growth of G. sulfurreducens can grow with oxygen as the sole terminal electron acceptor

Lin, Coppi, and Lovley. 2004. Geobacter sulfurreducens can grow with oxygen as a terminal electron acceptor. Appl. Environ. Microbiol. 70: (in press).

Growth

Acetate

Growth of Geobacter on oxygen provides an explanation for how Geobacter survives in low numbers in aerobic subsurface environments and then rapidly responds to the development of anaerobic conditions when uranium bioremediation is initiated.

Knocking out the cytochrome oxidase genes inhibits growth of G. sulfurreducens on oxygen

• Cytochrome oxidase iscomprised of four genes,ORFs 374, 376, 378, and 380.

• Mutant is a deletion of 374, 376and replaced with antibioticresistance cassette.

• Mutant does not grow with O2 but still can consumeO2.

• Implications:-Terminal oxidase is

responsible for growth with low % O2.

- Inactivation of terminal oxidase does not affect the activity ofoxidative stress enzymes.

Wild Type Growth

Mutant Growth


• Elucidation of novel mechanism for Geobacter species to find andaccess 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�(III)Oxide

Fe�(III)Oxide

Fe(II)

Geobacter Specifically ExpressesFlagella when only Fe(III) Oxide is Available as an Electron Acceptor

Geobacter follows Fe(II) Gradientto Locate Fe(III) Oxides

Geobacter May use Flagella to Make Initial Contact with Fe(III)

Fe�(III)Oxide

Fe�(III)Oxide

Geobacter uses Pili to “Twitch” AlongSediment 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


• Elucidation of genes encoding for key respiratory proteins

Provides molecular targets for estimating rates of metal reduction in the subsurface.

O2, NO3, SO4

Reduction at inner membraneor in the cytoplasm

Fe(III) Fe(II)

Electron Transfer to Extracellular Electron Acceptors Such as Metals and Electrodes is Fundamentally Different than the

Reduction of Commonly Considered Soluble Electron Acceptors

???

0

20

40

60

80

100

120

140

160

0 1000 2000 3000 4000 5000 6000Total # of ORFs in each Organism

GS

AF

PA

AA

r2= 0.70p< 0.01

Geobacter sulfurreducens has a unusually highpercentage of genes devoted to electron transportmany of which encode for c-type cytochromes

Fe(III)

NADH

NAD

DH

Fe(III)

omcB

OmpAOmcD

MacA

MQred

PppA,C,D

,E OmcEFe(II)

MQox OmpB

Model for Electron Transfer to Model for Electron Transfer to Fe(III) in Fe(III) in GeobacterGeobacter

OmcB OmcB but not but not OmcC OmcC is Requiredis Requiredfor Fe(III)Reductionfor Fe(III)Reduction

0

10

20

30

40

50

60

0 20 40 60 80 100 120

Hours

Fe(I

I) m

M

Wild type

∆omcC:: kan

∆omcB::cam

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.

R2 = 0.9355

0.00E+00

1.00E+05

2.00E+05

3.00E+05

4.00E+05

5.00E+05

0.1 0.15 0.2 0.25 0.3 0.35Fe(III) reduction rate (mmol electrons/mg protein/h)

omcB

tran

scri

pts

(cop

y nu

mbe

rs/ µ

g to

tal R

NA

)

Direct Correlation Between levels of omcB mRNA and Rates of Fe(III) Reduction in Acetate-Limited Chemostats of Geobacter sulfurreducens


• 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.

ENVIRONMENTALENVIRONMENTALSTIMULISTIMULI

Signals

OTHER GLOBAL REGULATORS

SIGMA FACTORS TWO COMPONENT SYSTEMS

RESPONSE

REGULATORY CASCADESTO CONTROL TRANSCRIPTION

Differentialgene expression

RpoS RpoE Histidine kinasesResponse regulators

RelA Fur

ELECTRON TRANSFER

Wildtype RpoS mutant

RpoS

-10-35

RNA pol

TARGET GENE

EXPRESION

PROTEOMICS: C. Giometti

MICROARRAYS

• Stationary phase may more closely represent physiological state in subsurfaceenvironments 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 Defining the RpoS regulon RpoS regulon in in G. G. sulfurreducenssulfurreducens

B. MetheGenetics: C. Nunez

RpoE

-10-35

RNA pol

TARGET GENE

EXPRESIONMICROARRAYS

• RpoE RpoE REGULON:REGULON:1. Cytochrome genes (7) and cytochrome biogenesis

genes involved in Fe(III) reduction2. Oxidative stress regulon different from RpoS3. Biofilm metabolism and development

4. Biofilm electron transfer via H2

Defining the Defining the RpoE regulon RpoE regulon in in G. G. sulfurreducenssulfurreducens

An rpoE mutation affects the expression of at least 200

other genes

50 µm

RpoE-WT

Target geneRNA pol ppGpp

RelAppGpp

Slower Growth (protein synthesis, nutrient transport)Increased resistance to Oxidative Stress ResistanceIncreased production of cytochromes for Fe(III) Reduction

Starvation

GTP

Rel Rel A Plays an Important Role in Regulating Growth and A Plays an Important Role in Regulating Growth and Metabolism in Metabolism in Geobacter sulfurreducensGeobacter sulfurreducens underunder

Environmentally Relevant ConditionsEnvironmentally Relevant Conditions

Phenotype of relA Mutant• Increase growth under nutrient limitation• Decrease growth in presence of oxygen• Upregulation of genes involved in:

protein biosynthesiscell divisiontransport

• Downregulation of genes for:stress responsesignal transductioninsoluble Fe(III) reduction

Microarray Results Comparing Wild Type to the Microarray Results Comparing Wild Type to the furfur Mutant Mutant

Regulators

Metabolism

Metal Uptake

Cytochromes

Unknown Proteins

Up regulation of 9 regulatory genes was found, including dtx, another iron regulated repressor.

Up regulation of 15 genes involved in metabolism including HydB, the hydrogenase responsible for hydrogen dependant growth.

Up regulation of 16 possible metal uptake genes was found, including FeoB,a ferrous iron cytoplasmic membrane transporter.

Up regulation of 7 cytochrome genes was found, including OmcB and OmcD.

Up regulation of 32 genes with an unknown function.

WT 941 Mutant

1-D and 2-D SDS-PAGE stained for c-type cytochromes (heme)

Knocking out a Histidine Kinase Sensor Inhibits Cytochrome Production

WT 941 Mutant

0

2

4

6

8

0

Fe(II

) mM

Succ

inat

e (m

M)

15

Time (h)Fe(III) pulse

Fe pulsecontrol

frdCAB mRNA levels

0

10

8

12

14

5 10 20 25 30

succinateFe(II)

Fe(III)-Specific Regulation of Fumarate Respiration

Abraham Esteve-Núñez, Cinthia Núñez and Derek R. Lovley. 2004. J. Bacteriol. (in press).


• Elucidation of systems regulating:growth under slow, environmentally relevant conditionsresponse to environmental stressresponse to nutrient limitation

Provides information necessary to interpret the in situ metabolic state of Geobacter species in the subsurface.


• 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.



• Development of an in silico model that can:

-- Predict the response of Geobacter to different environmentalconditions including strategies for manipulating the environment topromote bioremediation

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

Contributions of Iterative Contributions of Iterative In In SilicoSilico Model Building Model Building to Understanding of the Environmental Responses to Understanding of the Environmental Responses

ofof Geobacter Geobacter

Genome SequenceInformation

In vitro/in vivo characteristics

Added NetworkFunction

Prediction

Revised GenomeAnnotations

RefinementInferred Fitness &

Capabilities

ComputationalExperiment

BiochemicalExperiment

in silico-basedhypothesis

in silicoCellular Models

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

Genome-scale model of G. sulfurreducens

Given a limited number of constraints, growth rate, yield, andflux through metabolic network can be quantitatively predicted

Predicted growth rate vs. observed Flux (example)

Fe�(III)Oxide

)

Fe(II)

Microbially ProducedReduced Shuttle

OxidizedShuttle

Release of Shuttle Fe(II)

Fe(III)

Fe�(III)Oxide

Fe(III)

C <

C

00.02

0.040.06

0.08

0.1

MQ

N3

MQ

N4

MQ

N5

MQ

N6

MQ

N7

MQ

N8

MQ

N9

00.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Normalized Growth Rate

Menaquinone Secretion Rate (mmol/gdw hr)

Number of Units

Energetics of Menaquinone Secretion (10 mmol/gdw hr Acetate Uptake)

0.9-10.8-0.90.7-0.80.6-0.70.5-0.60.4-0.50.3-0.40.2-0.30.1-0.20-0.1

Analysis of Metabolic Cost to Release a Quinone-Based Electron Shuttle

Simulations carried out for varying

• Quinone secretion rates

• Different Sized Molecules

Significant growth rate reduction due to both ATP requirements and carbon requirements for shuttle synthesis

Provides likely explanation for the predominance Geobacterover Geothrix in subsurface environments



• Discovery of significant similarities in genomes of as-yet-unculturedGeobacter 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 Medium with Aquifer Clay Fraction Serving as Fe(III) Source

Phase Contrast Micrograph of CellsAmongst Sediment Clay FractionIn Ground-Water Amended Medium

orf1 orf2 omcB orf3 orf4 omcC

ATP binding permease

GDEF protein

Stress response protein

Metal binding protein

adenosyltransferase aminotransferase periplasmic protein

ABC transporter

oxidoreductase transporter

transporter inner membrane protein

gyrB gyrA gspA 16S trna 23S trna 5S

dnaJ dnaN recF dnaK

lexA dinP

RNA ligase

hydrolase

tbB tolQ hrcA grpE

histidine kinase response regulator

protophoryin oxidase marR

BAC clone from as-yet-uncultured Geobacter from subsurfacesediments containsomcB, a gene for anouter-membranecytochrome requiredfor Fe(III) reductionin G. sulfurreducensin the same geneorganization as seen inG. sulfurreducens

ompB ompB lipoprotein

tetR

hlyD

acrB

membraneprotein

sensory boxhistidine kinase

dehydropantoatereductase

LuxR

effluxprotein

effluxprotein

effluxprotein

effluxprotein

responseregulator

tetR

fibronectin

moeA

drug resistance

drug resistance

Map of BAC 109

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

thiamine-phosphatepyrophosphorylase selenide dikinase

phosphoribosylaminoimidazole-carboxamideformyltransferase resB

ppcA

glycosyltransferaselipopolysaccharide transporter

glycosyltransferase

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

ribonuclease hydrolase tetrapyrrole methylase

alpha amylase

BAC 83

G. sulfurreducens

63.6%

General Secretion Pathway

gspL

65.8%

52.4%

66.4%

67% 56%

67.2% 63.8%73% 62.8%

gspK gspJ

gspI gspH gspG gspF gspDgspE

hypothetical

gspL gspK gspJ

gspI gspH gspG gspF gspDgspE

hypothetical

% Similarity

BAC 84

G. sulfurreducensflgB

61.9%

Flagella

flgEflgD

flgC fliE fliF fliG fliH fliJ

fliL fliM flhBfliN fliP fliQ fliR

flgB flgC fliE fliF fliG fliH fliJ

flgEflgD fliL fliM(end of Bac)

65%

66.3% 63.3% 63.2%

63% 71.9% 67% 66.2% 66%

52.1%

% Similarity

pilB pilT pilC pilin domain

pilin domainpilCpilTpilB

BAC 96

G. sulfurreducens

62.2%56.5%60.5%62.6%

Pilin

% Similarity

BAC 82

G. sulfurreducenssubunit

1subunit

4subunit

2subunit

3

subunit1

subunit4

subunit2

subunit3

Cytochrome Oxidase

% Similarity 64.5% 60% 62.5% 66.7%

BAC 81

G. sulfurreducens

56% 66.2% 58.2% 64%

Maturationprotein

Largesubunit

CompetenceF

Hydrogenase A

Smallsubunit

Maturationprotein

Largesubunit

CompetenceF

Smallsubunit

% Similarity

BAC 88

G. sulfurreducensSmall

subunit

63.8% 68.5% 61% 63.7%

Membraneprotein

Largesubunit

FeSsubunit

Hydrogenase B

Smallsubunit

Membraneprotein

Largesubunit

FeSsubunit

% Similarity

fdhA fdhB fdhC fdhDBAC 147

G. sulfurreducensfdhA fdhB fdhC fdhD

60% 61.4% 63.3% 50%

Formate Dehydrogenase

BAC 74

G. sulfurreducensnifHnifDnifK

61.9%63%62%

Nitrogen Fixation Genes

nifHnifDnifK

% Similarity

EnvironmentalGenomic DNA of“As-Yet-Uncultured”Geobacters

Geobacter Genetic Potential

Novel CulturingStrategies to IsolateEnvironmentally RelevantGeobacters

Previously Cultured Geobacters

Analysis of Gene Expression in Relevant Environments with Environmental Genome Arraysand Proteomics

Functional GenomicsElucidation of Regulatory SystemsAnalysis of Gene ExpressionPhysiological, Biochemical Studies

In Silico Model of Cell Function

In Silico and Conceptual Models for OptimizingUranium Bioremediation and Electrical Energy Harvesting

Genome Sequencing

Application of Environmental Genomics and Systems Biology to UraApplication of Environmental Genomics and Systems Biology to Uranium nium Bioremediation and Harvesting Electricity from Waste Organic MatBioremediation and Harvesting Electricity from Waste Organic Matterter

nifD and recA expression in Acetate-amended and Control Subsurface Sediments before and after adding 100 µM NH4Cl

1

10

100

1000

10 4

10 5

10 6

10 7

acetate (nif)control (nif)acetate (rec)control (rec)

mR

NA

exp

ress

ion

per µ

g of

tota

l RN

A

Day 0 Day 1 Day 2

Geobacter Genes Upregulated During Growth on Electrodes

heat shock protein, Hsp20 familydnaJ domain proteinheat shock protein, Hsp20 familyheat shock protein, Hsp20 familyheat shock protein, Hsp20 familycytochrome c family proteinhypothetical proteinNOL1/NOP2/sun family proteinconserved domain proteinC4-dicarboxylate transporter, anaerobiccytochrome c family protein, putativecytochrome c family proteinClpB proteinNHL repeat domain proteinmetal ion efflux outer memb. prot. family, put.hypothetical proteinhypothetical proteinABC transporter, permease proteintranscriptional regulator, MerR familyhypothetical protein

Geobacter Genes Upregulated During Growth on Electrodes

heat shock protein, Hsp20 familydnaJ domain proteinheat shock protein, Hsp20 familyheat shock protein, Hsp20 familyheat shock protein, Hsp20 familycytochrome c family proteinhypothetical proteinNOL1/NOP2/sun family proteinconserved domain proteinC4-dicarboxylate transporter, anaerobiccytochrome c family protein, putativecytochrome c family proteinClpB proteinNHL repeat domain proteinmetal ion efflux outer memb. prot. family, put.hypothetical proteinhypothetical proteinABC transporter, permease proteintranscriptional regulator, MerR familyhypothetical protein

OmcD

0

0.2

0.4

0.6

0.8

1

0 50 100 150 200 250

Cur

rent

(mA

)

Hours

Wild type

OmcD Mutant-Complemented

OmcD Mutant

Effect of Deletion Mutation in omcD onCurrent Production

Support of NABIR by GTL in the Future

• Use of molecular techniques to assess in situ rates ofmetal reduction.

• Whole-genome analysis of in situ gene expression todetermine the in situ metabolic state of microorganismsduring uranium bioremediation which will help directimplementation of bioremediation strategies.

• Coupling in silico microbial models with geochemical andhydrological models to accurately predict the rate andextent of bioremediation in diverse environments undervarious bioremediation strategies.

eobacter Project · GeoBattery e Bond, Holmes, Tender, and Lovley. 2002. Science 295:483-485...

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Transcript of eobacter Project · GeoBattery e Bond, Holmes, Tender, and Lovley. 2002. Science 295:483-485...