Recent advances in bacterial community involvement in mercury transformations in theinvolvement in mercury transformations in the environment

Anthony V. Palumbo*

Jerry M. Parks, Alexander Johs, Mircea Podar, Jennifer J. Mosher,Jerry M. Parks, Alexander Johs, Mircea Podar, Jennifer J. Mosher, Dwayne A. Elias, Romain Bridou, Richard A. Hurt Jr., Steven D. Smith, Stephen J. Tomanicek, Yun Qian, Steven D. Brown, Craig C. Brandt, Tatiana A. Vishnivetskaya, Baohua. Gu, Scott C. Brooks, Jeremy C. Smith, Judy D. Wall, and Liyuan Liang**

* Biosciences Division Oak Ridge National Laboratory

**The Mercury SFA is led by L LiangThe Mercury SFA is led by L. Liang,Environmental Sciences Division, ORNLwith University of Missouri as a partner EFPC

Mercury is an important pollutant

• Microbial transformations of mercury as itmercury as it moves though the geochemical cycle are critical in itsare critical in its toxicity

Mercury sources range from natural emissions, to emissions from coal fired power plants andto emissions from coal fired power plants, and releases from industrial sources

• Mercury is a wide spread pollutant a at low levels duepollutant a at low levels due to atmospheric deposition

• Hot spots are usually yassociated with industrial point sources.

Bioaccumulation can exacerbate the problemthe problem

Predator fish 10-100 million x

Prey fish, 1-10 million x

Zooplankton, 20,000 – 100,000 x

Phytoplankton, 10,000 x

Water, 1x methylmercury

Hg concentrations in wildlife

www.briloon.org

Hg cycling is complex and there is a microbial involvement in methylation and demethylation

Hg(II) Wet and Dry DepositionV l tili ti

Emission & Transport

OxidationHg(II)Hg(0)

y y

Inflow&run off

Volatilization

Hg(0)

Hg(II)-LMeHg

Hg(II) L

Hg(II)-L

Settling/Resuspensiondiffusion

Hg(0)

BioaccumulationHg(II) LMeHg

Bioaccumulation

Methylmercury research timeline 

1956 Minamata disease discovered and in 1968 officially1956 – Minamata disease discovered and in 1968 officially recognized that disease was caused by the consumption of seafood contaminated by methylmercury compounds.methylmercury compounds.

1967 to 1969 – First reports that methylation of mercury may be bacterial in origin, and that anaerobic 

ff hi i (ecosystems can effect this reaction (Jensen and Jernelov, 1967, 1968, 1969 Nature, 223: 753‐754; Wood et al. 1968 Nature, 220:173)

Minamata disease: a neurological syndrome caused by severe mercury poisoning.

1974 –Methyl‐B12 compounds are likely the agent for transferring CH3

‐ to Hg.  (Wood, JM. Science, 183:1049.)

1994 R h h th t th l f ti1994 – Researchers show that methylmercury formation can be an enzymatic process involving  cobalt. (Choi et al, AEM, 60(4):1342)

2003 – Work contradicts the 1994 study, questioning the role of B12. (Ekstrom et al. AEM, 69(9):5414)

The ORNL/DOE Mercury Science Focus area examines a pollutant important to DOEexamines a pollutant important to DOE • Reductions of mercury

i t i EFPK h2.0

input in EFPK have not led to decreases in Hg in FishTh US D t t f 1 4

1.6

1.8

er)

Water

• The US Department of Energy has been funding a research program at the Oak

1.0

1.2

1.4

sh),

礸/

L (

wa

te

program at the Oak Ridge National Laboratory focused on increasing the 0 4

0.6

0.8

Hg

, 礸

/g

(fi

sFish

increasing the understanding of mercury transformations in the 0.0

0.2

0.4

1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008environment. 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008

The Hg SFA strategy for understanding contaminant transformation and behavior

Microbial andgenetic controls

Fundamental ratesand mechanisms

Molecular structure and simulations

Field biogeochemistry

Hg2+ + 2e- = Hg(0)

catalytic domain

NmerA1 ) 20

1025

1 ) 20

1025

nten

sity

(a.u

.)

NmerA

MerA core

N-terminus

C-terminus

CYS 11

CYS 14

CYS 136

CYS 141

CYS 558

CYS 559

(Luther et al. 1999) 0.01 0.1 1 10 100 1000

K DO

M' (

L kg

- 11010

1015

1020

Thiol-like binding

Carboxyl bindingCarboxyl bindingCarboxyl binding

0.01 0.1 1 10 100 1000

K DO

M' (

L kg

- 11010

1015

1020

Thiol-like binding

Carboxyl bindingCarboxyl bindingCarboxyl binding1000 1200 1400 1600 1800

In

Haitzer et al. (2003)

Transformation in field Speciation & mechanisms Molecular dynamics

Sediment-water interfaceSpecies/ abundance Microbial communities

Coupled microbialand geochemicalreactions

Molecular level understanding

of contaminant associationand reaction

Reaction mechanisms and kinetics at groundwater–surface water interface

Site Site biogeochemical processes and biogeochemical processes and microcosm studiesmicrocosm studies

Site Investigations• Chemical, physical, microbial data relevant

to Hg transformations • Hg reactivity

Hg Biogeochemistry

Hg0 Hg(II) MeHgoxid. meth.

Hg0, MeHgvolatilization

Hg Biogeochemistry

Hg0 Hg(II) MeHgoxid. meth.

Hg0 Hg(II) MeHgHg0 Hg(II) MeHgoxid. meth.

Hg0, MeHgvolatilization

Microelectrodes

Field measurementsField measurements

• Hg reactivity• Correlating Hg reactivity with methylation

potential Methylation Bioassay

MeHg,Hg0, Hg(II)Diffusion

Particle boundMeHg, Hg(II)

Settling,Resuspension

g g( ) gred. demeth.

Hg0 Hg(II) MeHgoxid.

red.

meth.

demeth.

MeHg,Hg0, Hg(II)Diffusion

Particle boundMeHg, Hg(II)

Settling,Resuspension

g g( ) gred. demeth.

g g( ) gg g( ) gred. demeth.

Hg0 Hg(II) MeHgoxid.

red.

meth.

demeth.Hg0 Hg(II) MeHgHg0 Hg(II) MeHg

oxid.

red.

meth.

demeth. Flux chambers

(Luther et al., 1999)

y y• Hg speciation and methylation rate

Microcosm Studies• Methylation/ demethylation in ecologically

i t t t

BurialBurial

(Menheer, 2004)

intact system Enclosure Studies

• key variables on net methylation in-stream Data Analysis Geochemical

Complementary laboratory microcosms

Data Analysis, Geochemical Modeling, Site Conceptual and Numerical ModelFunctional gene

arraysGeochemical modeling

* Critical understanding of Hg flux biogeochemical controls

* Critical understanding of Hg flux biogeochemical controls-15

-10

-5

log

{HS

-}

MeHgS-

(MeHg)2S

flux, biogeochemical controls, and microbial determinantsflux, biogeochemical controls, and microbial determinants-20

-5.5 -4.5 -3.5 -2.5 -1.5

log {Cl-}

MeHgOH0 MeHgCl0

Fundamental mechanisms and transformations

Speciation and geochemical controls• Rates and mechanisms, oxidation/reduction• Single reactant to multi-component systems Cysteine

2MPA

g y• Hg-microbial cell interactions and Hg uptake

Roles of DOM and POM in Hg methylation, demethylation, and redox transformations

Glutathione

Penicillamine

• Specific moieties and functional groups

• EXAFS analysis, speciation and coordination chemistry

Glutathione

10-5

Hg-DOM(thiol)

• Species, models, and effects on bioavailability

Catalyzed surface and photochemical reactions

• Roles in Hg reactivity and demethylation60Initial Hg(II)=10 nM

10-20

10-15

10-10

Hg-DOM(carboxyl)Hg(OH)2

peci

es, m

ol/L

HgCl2

1

32

EFPC

* Critical understanding of dominant Hg

• Roles in Hg reactivity and demethylation• Sorbed species and reactions• Labeled stable isotope studies

20

40

with humicsHg(II) reduction

 redu

ced (%

)

1 2 3 4 5 6 7 8 9 1010-30

10-25

1054

Hg(

II) S

67

89 10 11

* Critical understanding of dominant Hg species, its bioavailability, and biogeochemical controls on rates and mechanisms of Hg methylation and d th l ti

0 3 6 90

20

Control blank

DOM in solution (mg/L)

Hg(II) pH

demethylationDOM in solution (mg/L)

Microbial and genetic controls on Hg methylation Verification and validation of hgcAB as a

di ti f H th l tipredictive measure of Hg-methylation potential Testing of predicted methylators and non-

methylators during growthDesulfovibrio africanus48h

y g g Elucidate the native biochemical role and

regulation of hgcAB• Determine the effect of geochemical factors on gene 0.8

1.0

1.2

o

Sigmoidal cells Persister cells 201MeHg produced

140

160

180

200

) g gregulatory networks for mercury methylation

• Comparative gene expression, mutagenesis, and complementation

Develop and utilize “universal primers” for0.4

0.6

0.8

Mor

phot

ype

Rat

io

60

80

100

120

140

Met

hyl-20

1 Hg

(ng/

L)

235

Develop and utilize universal primers for determination of Hg-methylation potential in any environment.

Examine relationships among community0 10 20 30 40 50

0.0

0.2

Time (hrs)

0

20

40

1

1.5

15

20

25

30

Hg (m

g L)

bund

ance

Desulfobulbus

Desulfonema

Examine relationships among community structure, geochemical conditions, and methyl Hg production in sediments globally• hgcAB biomarker

0

0.5

0

5

10

15

EFK23 EFK13 EFK6

Me

% Ab

MeHg • Pyrosequencing and metagenomics*Critical understanding of the genetic basis of the methylation and demethylation processes and the geochemical controlsEFK23 EFK13 EFK6 processes and the geochemical controls on microbial transformation.

Molecular structure, dynamics and simulations

Establish biochemical pathways in bacterial demethylation

Obtain structure of protein/protein and protein/DNA complexesand protein/DNA complexes• Apply small angle neutron scattering to

reveal structure-function relationships Reveal enzymatic mechanisms to Reveal enzymatic mechanisms to

understand the processes of demethylation and reduction• Use quantum mechanical/molecularUse quantum mechanical/molecular

mechanical simulations

*Critical understanding of the gbiochemical and biophysical mechanisms in Hg transformation (demethylation and methylation) by developingand methylation) by developing and validating subcellular models and investigation of structure-function relationships

Barkay, T., S.M. Miller, and A.O. Summers: FEMS Microbiol Rev, 2003. 27(2-3): p. 355-84.

Two parts of the puzzle: Community composition (Tasks 1 and 3) and Gene identity (Tasks 3 and 4)(Tasks 1 and 3) and Gene identity (Tasks 3 and 4) Community Studies• Mosher, J. J., T. A. Vishnivetskaya, D. A. Elias, M. Podar, S. C. Brooks, S. D. Brown, C. C. Brandt and A. V. Palumbo.

2012 Characterization of the Deltaproteobacteria in contaminated and uncontaminated surface stream sediments and2012. Characterization of the Deltaproteobacteria in contaminated and uncontaminated surface stream sediments and identification of potential mercury methylators. Aquatic Microbial Ecology. 66:271-282.

• Porat, I., Tatiana A. Vishnivetskaya, Jennifer J. Mosher, Craig C. Brandt, Zamin K. Yang, Scott C. Brooks, Liyuan, Liang, Meghan. M. Drake, Mircea Podar, Steven D. Brown, and Anthony V. Palumbo. 2010. Characterization of the Archaelcommunity in contaminated and uncontaminated surface stream sediments. Microbial Ecology. 60: 784-795. doi: 10 1007/s00248-010-9734-210.1007/s00248-010-9734-2

• Vishnivetskaya, T. A., J. J. Mosher, A. V. Palumbo, M. Podar, S. D. Brown, S. C. Brooks, Z. K. Yang, B. Gu, G. Southworth, M. M. Drake, C. C. Gilmour, J. D. Wall, C. C. Brandt and D. A. Elias. 2011. Mercury and other heavy metals influence bacterial community structure in contaminated Tennessee streams. Appl. Environ. Microbiol. 77:302-311.

G Id titGene Identity• Gilmour, C. C., D. A. Elias, A. M. Kucken, S. D. Brown, A. V. Palumbo, C. S. Schadt, and J. D. Wall. 2011. The sulfate-

reducing bacterium Desulfovibrio desulfuricans ND132 as a model for understanding bacterial mercury methylation. Appl. Environ. Microbiol. 77:3938-3951.

• Brown Steven D J D Wall A M Kucken C C Gilmour M Podar C C Brandt H Teshima C S Han J C Detter M• Brown, Steven D., J. D. Wall, A. M. Kucken, C. C. Gilmour, M. Podar, C. C. Brandt, H. Teshima, C. S. Han, J. C. Detter, M. L. Land, S. Lucas, J. Han, L. Pennacchio, M. Nolan, S. Pitluck, T. Woyke, L. A Goodwin, A. V. Palumbo, and D. A. Elias. 2011. Genome sequence of mercury-methylating and pleomorphic Desulfovibrio africanus strain Walvis Bay. Journal of Bacteriology 193: 4037-4038.

• Brown, Steven D., C. C. Gilmour, A. M. Kucken, J. D. Wall, D. A. Elias, C. C. Brandt, M. Podar, O. Chertkov, B. Held, D. C. Bruce J C Detter R Tapia C S Han L A Goodwin J-F Chen S Pitluck T Woyke N Mikhailova N N Ivanova JBruce, J. C. Detter, R. Tapia, C. S. Han, L. A Goodwin, J F Chen, S. Pitluck, T. Woyke, N. Mikhailova, N. N. Ivanova, J. Han, S. Lucas, A. L. Lapidus, M. L. Land, L. J. Hauser, A. V. Palumbo. 2011. Genome sequence of mercury-methylatingDesulfovibrio desulfuricans ND132. Journal of Bacteriology 193:2078-2079.

• Parks, J.M., A. Johs, M. Podar, R. Bridou, R.A. Hurt, S.D. Smith, S.J. Tomanicek, Y. Qian, S.D. Brown, C.C. Brandt, A.V. Palumbo, J.C. Smith, J.D. Wall, D.A. Elias and L. Liang. 2013. The genetic basis for bacterial mercury methylation Science (2013) 339:1332 1335bacterial mercury methylation. Science (2013) 339:1332-1335.

The study site included several streams in the Oak Ridge area including a control siteOak Ridge area including a control site

AA

There is a seasonal aspect to methylmercuryconcentrations that points to biologicalconcentrations that points to biological processes

700.45

60

0.35

0.4

40

50

(ng/

L)0.25

0.3

g (n

g/L)

20

30

Hg

(

0.15

0.2MeH

g

0

10

0

0.05

0.1

04914192429

EFK km (values decrease downstream)

0

DHg - Dec 2007 DHg - June 2008 DMeHg - Dec 2007 DMeHg - June 2008

Phylogenetic analysis The primary method of phylogenetic analysis was via PCR amplification and 454

Pyrosequencing The hypervariable V4 region (~290 bp) of the 16S rRNA gene was amplified from The hypervariable V4 region (~290 bp) of the 16S rRNA gene was amplified from

the total community genomic DNA using the high fidelity AccuPrime Pfx DNA polymerase (Invitrogen, Carlsbad, CA) and specially designed primers.

The primers in addition to the 16S rRNA priming region contain sequences The primers, in addition to the 16S rRNA priming region, contain sequences (adaptors) required for 454 FLX pyrosequencing, and the forward primer contains an additional short key (tag) sequence.so that 40 samples could be analyzed in one sequencing runsequencing run..

Raw 454 data (~180,000 Mb from whole plate: region A and B) were initially processed trough RDP pyrosequencing pipeline. During this process the sequences were sorted by tag sequence trimmed off the 16S primers and filtered out of low-were sorted by tag sequence, trimmed off the 16S primers and filtered out of low-quality sequences. From ~8,000 to ~12,000 high quality sequences by size 200-220 bp were obtained for each sample.

Samples taken over three quarterly sampling periods (36 samples) Samples taken over three quarterly sampling periods (36 samples). The Deltaproteobacteria are the group of bacteria that are generally

acknowledged to be involved in mercury methylation.

Relative numbers of Deltaproteobacteria in these surface sediments were lowthese surface sediments were low

DeltaFirmicutes

Control Site

Delta

Contaminated Site

The most common Deltaproteobacteria was DesulfobulbusDesulfobulbus

There were seasonal trends in community abundanceabundance

Desulfobulbus is considered a logical candidate as a methylator in this streamy

•There was a statisticallystatistically significant correlation of methyl-mercury y yconcentrations with several Deltaproteobacteria includingincluding Desulfobulbus spp., Desulfonema spp., and Desulfobaccasppspp.

Triplot of the redundancy analysis (RDA) for bacterial phyla from the 20 stream sediments samples at five sites located (BCK samples excluded) on or near the Oak Ridge Reservation with forward selection of predictor variables followed by Monte Carlo permutationspredictor variables followed by Monte Carlo permutations

Identifying the genes involved in mercury methylation required an integrated omics andmethylation required an integrated omics and biochemical approach• The Oak Ridge National Laboratory’s research team has

used comparative genomics in the context of biochemical pathways to identify key genes involved in mercurypathways to identify key genes involved in mercury methylation.

• The basis for this comparative analysis was the sequencing f l b t i bl f th l ti d thof several bacteria capable of methylation under the

leadership of the Oak Ridge National Laboratory’s research program. p g

Desulfovibrio africanus(SEM by Dwayne Elias,)

Geobacter chapelleii, 172Geobacter pelophilus, Dfr2Geobacter bremensis, Dfr1

Is there a genetic basis for bacterial Hg methylation

DesulfuromonalesGeobacter bremensis, Dfr1Geobacter lovleyi, SZ

Geobacter metallireducens, GS-15Geobacter grbiciae, TACP-2Geobacter hydrogenophilus, strain H4Geobacter sulfurreducens, PCADesulfuromonas palmitatis, SDBY1

Desulfuromonas chloroethenica, TT4BDesulfuromonas thiophila NZ27 (DSMZ 8987)

methylation

D lf b t l

Desulfuromonas thiophila, NZ27 (DSMZ 8987)Desulfuromonas acetoxidans, DSM 624

Desulfocapsa sulfexigens, SB164P1Desulfotalea psychrophila, LSv54

Desulforhopalus vacuolatus, ltk10Desulfobulbus propionicus, DSM 2032

Desulfosarcina variabilisDesulfococcus multivorans, DSM 2059

Non Hg methylator

Weak Hg methylator

Strong Hg methylator DesulfobacteralesDesulfofrigus oceanense, ASv26Desulfobacterium sp., BG33

Desulfobacterium autotrophicum, DSM 3382Desulfobacter vibrioformis, B54Desulfobacter species, 4ac11 DSM 2057

Desulfobacter species, 3ac10 DSM 2035Desulfobacter hydrogenophilus DSM 3380

Desulfobacter curvatus, DSM 3379

Strong Hg methylator

Myxococcales,

Desulfobacter sp., BG8Corallococcus coralloides, DSM 2259

Myxococcus xanthus, DSM 435 (Mx x1)Desulfovibrio dechloracetivorans, ATCC 700912

Desulfovibrio desulfuricans, ND132Desulfovibrio profundus, DSM 11384

Desulfovibrio sp., T2Desulfovibrio sp W3A

DesulfovibrionalesDesulfovibrio sp., W3A

Desulfovibrio gigasDesulfovibrio sp., X2

Desulfovibrio desulfuricans, DSM1926 El Agheila Z

Desulfovibrio desulfuricans , MB; ATCC27774Desulfovibrio desulfuricans, Essex 6; ATCC29577Desulfovibrio vulgaris, Hildenborough

Desulfovibrio africanus

g , gDesulfovibrio alaskensis, G20Desulfovibrio alaskensis, NCIMB 13491

Desulfomicrobium baculatum, DSM 4028TDesulfomicrobium orale, DSM12838

Shewanella algae, BryShewanella oneidensis, MR-1Shewanella putrefaciens, CN-32

0.10

Genomes plus chemical reasoning points to the genes

CH3− + Hg2+ CH3Hg+

The biochemical reasoning led to a focusThe biochemical reasoning led to a focus on finding a corrinoid protein that could be required for mercury methylation.

HgcA HgcB

Two Genes appear to be involved HgcA HgcB

The two proteins are present in the genomes of all known methylatingy gbacteria that have been sequenced, but absent in all known non-methylators!

2 e−

pMO4651 was electroporated into D. d lf i ND132 (U Mi i) d

SpecRpUC ori SpecRpUC ori

Deletion of hgcA and hgcB

desulfuricans ND132 (U. Missouri) and G. sulfurreducens PCA (ORNL). Cells were plated for isolated colonies, picked and grown in liquid and checked for a “double cross-over event”.

UppKanRpKanDown streamUp stream

pMO4651

UppKanRpKanDown streamUp stream

pMO4651

Once verified, cells were assayed for methylation at U. Missouri and ORNL (stable isotopes/ICP-MS).

GOI#1056 GOI#1057

Wild type gDNAGOI#1056 GOI#1057

Wild type gDNA

D. desulfuricans ND132

Mutant gDNA UppKanRpKan

D. desulfuricans ND132 and G. sulfurreducens PCAD. desulfuricans ND132

Mutant gDNA UppKanRpKan

D. desulfuricans ND132 and G. sulfurreducens PCA Southern verification

Wild type32P Southern Probe (PCR 1,  ~0.7 Kb)

GOI#1056 GOI#1057

Mutant constructUppKanR

Expected fragment = 3.8 Kb

pKan

1) Digest genomic DNA for wild type and mutant with restriction enzyme (ZraI)

UppKanR

Expected fragment = 7.0 Kb

pKan

1) Digest genomic DNA for wild‐type and mutant with restriction enzyme (ZraI), probe with PCR product, which binds and yield a unique band for each strain.

Deletion studies confirm the importance of both genes in two methylatorsgenes in two methylators Experiment

2500

2000

2500

L)

G. sulfurreducens1500

cury

(ng/

L

500

1000

Met

hylm

erc

0

500

CA

440

441

441

M

urre

duce

nsP

DG

SU

1 4

DG

SU

14

DG

SU

1440

/14

G. s

ulfu D

Proposed Hg methylation pathway

HCO-THF 10-formyl-THF

H+

H O

5,10-methenyl-THF

cyclohydrolase

• The diagrams shows– potential sources of C1

units entering the

formateATP, 2e-

CH+=THF

THF synthetase

H+, 2e-

H2O

serine hydroxymethyl

transferase

units entering the reductive acetyl-CoA pathway,

– methyl group transfer from CH H folate (CH THF

serineglycineCH2=THF

5,10-methylene-THF

dehydrogenase

transferasefrom CH3-H4folate (CH3-THF) to cob(I)alamin-HgcA, methylation of Hg(II) by HgcA, and

d ti t b(I) l iCH3-THF CH3-Co(III)-HgcA

Co(I)-HgcA2H+, 2e-

5,10-methylene-THF

reductase?

– reduction to cob(I)alamin-HgcA by HgcB.

• In the absence of Hg, the methyl group is Co(I) HgcA

HgcB

2H , 2eHgR2

methyl group is transferred to a different substrate, which may be a physiologically relevant metabolite Co-HgcA

CH3HgR

Rmetabolite.

Figure in Parks et al (2013)Figure in Parks et al. (2013) adapted from Choi et al

Critical points

• These analyses led to the discovery of a two-gene cluster which has been designated hgcAB.which has been designated hgcAB.

• The involvement of these bacteria in mercury methylation has been confirmed using molecular techniques, which show that d l ti f ith h lt th l tideletion of either gene halts mercury methylation.

• Also, this two gene cluster is present in some bacteria (e.g., specific methanogens and clostridia) that have not as of yetspecific methanogens and clostridia) that have not, as of yet, been shown to methylate mercury.

• Thus, the diversity of mercury methylators could be greater y y y gthan previously known.

What are the implications? This discovery will change how mercury research is performed globally.

Immediate Microbiology team work based on the discovery

V if th di t bilit f th l ti i• Verify the predictability of methylating organisms. 

– Until now, methylators in 1 phylum. We predict at least 3 diverse phyla (and likely more). Do the others make more methylmercury and/or make it faster? 

• Develop a biomarker for methylmercury generation

– How prevalent are these genes in the environment? 

W l t l th l ti t ti l ith i d i– We can correlate real methylation potential with gene expression and organism abundance. 

– We can directly determine geochemical factors influencing biological mercury methylation in any environmentmethylation in any environment.

Implication: Correlating gene, protein and organism abundances with methylmercuryformation rates and yields will lead to improved and more sensitive o a o a es a d y e ds ead o p o ed a d o e se s ebiogeochemical models!   Desulfovibrio cell pellet

grown with and without Hg

Proteobacteria Firmicutes Euryarchaeota

Known and Predicted Methylmercury-Producing Microorganisms in 4 Phyla

Desulfovibrio desulfuricans ND132 Desulfovibrio aespoeensis Aspo-2 Desulfovibrio africanus str. Walvis Bay Desulfomicrobium baculatum X Desulfonatronospira thiodismutans ASO3-1

Acetivibrio cellulolyticus CD2 Dehalobacter restrictus DSM 9455 Dehalobacter sp. CF Dehalobacter sp. DCA Dehalobacter sp. FTH1

yMethanofollis liminatans GKZPZ Methanoregula boonei 6A8 Methanoregula formicicum SMSP Methanomassiliicoccus luminyensis B10 Methanosphaerula palustris E1-9c

Desulfonatronum lacustre Z-7951 Desulfovibrio oxyclinae DSM 11498 Desulfovibrio africanus PCS Desulfovibrio longus DSM 6739 Desulfovibrio putealis DSM 16056 Desulfobulbus propionicus DSM 2032

Desulfitobacterium dehalogenans ATCC 51507 Desulfitobacterium dichloroeliminans LMG P-21439 Desulfitobacterium metallireducens DSM 15288 Desulfitobacterium PCE1 DSM 10344 Desulfosporosinus acidiphilus SJ4 Desulfosporosinus orientis DSM 765

Methanospirillum hungatei JF-1 Methanolobus tindarius DSM 2278 Methanomethylovorans hollandica DSM 15978 Methanolobus psychrophilus R15 Methanocella arvoryzae MRE50 RC-I M th ll l di l SANAEDesulfobulbus propionicus DSM 2032

Desulfobulbus mediterraneus DSM 13871 Desulfospira joergensenii DSM 10085 Desulfotignum phosphitoxidans FiPS-3 uncultured Desulfobacterium sp. Geobacter sulfurreducens PCA

Desulfosporosinus orientis DSM 765Desulfosporosinus sp. OT Desulfosporosinus youngiae DSM 17734 Ethanoligenens harbinense YUAN-3 Syntrophobotulus glycolicus DSM 8271 Dethiobacter alkaliphilus AHT 1

Methanocella paludicola SANAE

Chloroflexi Dehalococcoides mccartyi DCMB5

Geobacter sulfurreducens DL-1 KN400 Geobacter metallireducens GS-15 Geobacter metallireducens RCH3 Geobacter sp. daltonii FRC-32 Geobacter sp. M18

pClostridium termitidis CT1112 Acetonema longum DSM 6540

Geobacter sp. M21 Geobacter uraniireducens Rf4 Geobacter bemidjiensis Bem Geopsychrobacter electrodiphilus DSM 16401 Syntrophorhabdus aromaticivorans UI D lf il ti dj i DCB 1 DSM 6799

This list is updated regularly by the ONRL team http://www.esd.ornl.gov/programs/rsfa/index.shtm

Desulfomonile tiedjei DCB-1 DSM 6799 Syntrophus aciditrophicus SB delta proteobacterium MLMS-1 delta proteobacterium NaphS2 Deferrisoma camini S3R1

Questions remain that will guide future researchQuestions• Are other genes involved?• Are the predictions of other methylators robust? •What determines rates of methylation and demethylation in the environment?demethylation in the environment?

•What are the mercury update mechanisms and forms of mercury taken up?

Future Work • Examine mercury methylation mechanism using in vivo and in vitro studiesand in vitro studies

• Test predicted organisms for methylation activity • Examine phylogenetic and functional relationships of p y g pmercury methylation in field samples with regard to geochemistry 

What are the implications?

• Will likely change how mercury research is performed globally

• Mechanism is new chemistry, yopening up new areas of research

• Biomarker for methylmercury generationy y g• Improved and more sensitive biogeochemical models

Summary

• These findings lay the groundwork for additional research• These findings lay the groundwork for additional research that will increase our understanding of the transformation of mercury in the environment. An increased understanding

ld l d t h i di ticould lead to new approaches in mercury remediation.

More than just another gene discovery

Chemicali

Three-dimensional Structural

bi i f ti Genetics Microbiologyreasoning dimensional thinking bioinformatics Genetics Microbiology

THE TEAM THE TEAM

J M P k Al d J h Mi P d R i B id Ri h d A H t J St D S ith Jerry M. Parks, Alexander Johs, Mircea Podar, Romain Bridou, Richard A. Hurt Jr., Steven D. Smith, Stephen J. Tomanicek, Steven D. Brown, Craig C. Brandt, Anthony V. Palumbo, Jeremy C. Smith,

Judy D. Wall, Dwayne Elias and Liyuan Liang

Acknowledgements

• This research was supported by the U S Department of• This research was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, through the Mercury Scientific F A t O k Rid N ti l L b t (ORNL)Focus Area at Oak Ridge National Laboratory (ORNL). ORNL is managed by UT Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.

• We thank S. M. Miller, C. C. Gilmour and T. Barkay for perceptive discussions on the chemistry, microbiology, and historical aspects of mercury methylationhistorical aspects of mercury methylation