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Recent advances in bacterial community ... Advances on Bacterial Community... · 2013-06-08 ·...
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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
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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
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Time (hrs)
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40
1
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20
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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
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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
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CA
440
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M
urre
duce
nsP
DG
SU
1 4
DG
SU
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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