Progress in Understanding Hg Transformations: From ... · 1 Managed by UT-Battelle for the U.S. Department of Energy Progress in Understanding Hg Transformations: From Molecular Modeling

1 Managed by UT-Battelle for the U.S. Department of Energy

Progress in Understanding Hg Transformations: From Molecular Modeling

to Microbial Community Dynamics

ORNL ERSP Science Focus Area (SFA)

SBR Annual PI Meeting, March 29-31, 2010


High mercury concentrations in biota •  High Hg(II) concentrations complexes in shallow

soils since 127,000 kg released via spills •  Oak Ridge environment: strong groundwater

interactions (>50” rainfall/yr) •  Hg exceeds regulatory limits

•  TN recently lowered Hg level that triggers an advisory

•  EPA concern for ecological risks

•  Methylmercury; •  is biomagnified up the food chain •  crosses the blood-brain barrier •  Poisoning irreversible

Elimination of inorganic Hg inputs not possible; alternative strategies that reduce methylation in-situ may be the only way to reach fish concentration targets

Basic research needed on mercury methylation/demethylation at sediment-water interface and particularly what limits methylmercury production EFPC downstream of Y-12


Systems model to address knowledge gaps for Hg transformations

Critical gaps in scientific understanding:

 Oxidation, reduction, and species transformation

 Dominant chemical species and bioavailability

 Abiotic /biotic methylation and demethylation

 Biochemical pathways for methylation and demethylation

 Coupled biogeochemical reactions

 Surface catalyzed and photochemical reactions

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Strategy for understanding contaminant transformation and environmental behavior

Microbiology and genetics

Rates and mechanisms

Transformation in field Speciation & mechanisms Molecular dynamics

Sediment-water interface Species/ abundance Microbial communities

Coupled microbial and geochemical reactions

Molecular understanding of contaminant association

and reaction

Molecular structure

catalytic domain

Field & geochemistry

(Luther et al. 1999)

Hg2+ + 2e- = Hg(0)

Haitzer et al. (2003)

New science: fundamental understanding of contaminant transformation


Mercury contamination across the Oak Ridge Reservation

Y-12

ORNL

ETTP

— Hg in water, sediment and biota in streams

Industrial use areas Hg contaminated soil, buildings, storm drain network, sediments, ground and surface water

Source Areas


Site geochemistry and influence on microbial community HCK20.6 EFK23.4 EFK13.8 EFK6.3 BCK12.3 WCK3.9

Hg dissolved (ng/L) 0.6-2.3 54.6-138.3 14.1-82.0 13.3-40.1 3.2-5.6 0.0-11.3 Hg sediment (ng/mg) 0.0-0.1 30.4-47.1 11.9-17.7 10.6-20.2 1.4-1.8 2.5-15.1 MeHg dissolved (ng/L)1 0.0-0.1 0.2-0.4 0.4-0.8 0.5-2.5 0.0-0.1 0.0-0.9 U (µg/L) 0.2-6.9 0.8-7.4 6.0-7.3 3.7-10.0 175.0-268.4 0.2-6.9 NO3

- (mg/L) 0-0.2 0.1-0.4 0.0-0.3 0.4-0.8 13.3-18.5 0.0-0.5 -pH, DIC, DOC, sulfate, sulfide (<0.15 µM) similar at all sites

Y12 Plant May 2008 July 2008

Vishnivetskaya et al., submitted ISME 2010.


Groundwater enrichment (Propionate + sulfate)

Five isolates obtained; Clostridium sp. ZP3 99% Coprococcus sp. 99% Desulfovibrio magneticus RS-1* 98% Clostridium sp. CITR8 98%

• Cultivation for Hg(II) methylation, reduction and MeHg demethylation.

J. Mosher, manuscript in preparation

pRD

A 2

14.

2%

-1.0 1.0

-0.8 1.0

Unclassified Deltaproteobacteria Unclassified

Syntrophobacteraceae

Geobacter

Desulfuromonas

Desulfonema

Desulfobulbus

Desulfocapsa

Anaeromyxobacter

Byssovorax

U

Turbidity

MeHg

Date 6.0%

pRDA 1 19.3%

Autocorrelations: U: Ba, Cl, Mg, Na, NO3, Sr, DIC

Turbidity: None MeHg: Mn 55

EFK13.8 EFK23.4 BCK12.3 HCK20.6 EFK6.3 WCK3.9

May 2008 July 2008

-Continuing to determine specific influences; • microbial community with revised functional

gene arrays, qPCR (merA,B), metagenome from high Hg methylation area.

• Planned microcosms; Hg(II), MeHg, U(VI), NO3


What are the predominant forms of Hg and MeHg in-situ?

Amount of particulate (complexed) Hg(II) increases with increasing distance from source.

**similar to the decrease in Hg(II) and concomitant increase in MeHg.

(Miller et al., 2009. Eviron. Sci. Technol. 43:8548-53)

Suwannee River DOM amendment


Modeling indicates MeHg is predominantly complexed with dissolved organic matter (DOM) and high log K suggests high complex stability once formed.

(Dong et al., 2010. Environ. Chem. 7:94-102.)

X-ray micro-images and elemental distribution of a phytoplankton from EFK show cellular association with several elements including Hg (K. Kemner, ANL).

Is NOM/DOM complexed Hg cell associated? Is NOM/DOM complexed Hg bioavailable? Does it inhibit methylation?


Is complexed DOM/NOM complexed Hg(II) bioavailable for methylation?

MeHg production during D. desulfuricans ND132 growth in the absence of complexants

D. desulfuricans ND132 cells grown in the absence of possible Hg complexants

•  +/- 10 mg/L bulk Suwanee River DOM

~ 50 ng/L MeHg ~ 60 ng/L MeHg

~ 200 ng/L MeHg

~ 650 ng/L MeHg

P < 0.01


Demethylation in sterile medium

Demethylation by cultures

MeHg production by cultures

pyruvate/fumarate medium

Demethylation in sterile medium

Demethylation by cultures

MeHg production by cultures

lactate/sulfate medium

7% Hg(II) methylation 57% MeHg demethylation in culture 20% MeHg demethylation in medium 1% Hg(II) reduction in culture 1% Hg(II) reduction in medium

30% Hg(II) methylation 20% MeHg demethylation in culture 20% MeHg demethylation in medium 1% Hg(II) reduction in culture 1% Hg(II) reduction in medium

Gilmour et al., 2010. Manuscript in preparation


D. desulfuricans ND132 genome now sequenced

Sequencing of ND132 gDNA is in the closing stages at JGI:

• 1 scaffold + 35 contigs. • Genome size=3.8Mb • 65.2% G+C • 3478 candidate protein-encoding genes

NO mer genes!!

Potential Consequences: Novel Hg transport into the cell? Method of Hg(II) methylation? Method of MeHg demethylation? Is there transcriptional regulation?


Genetic and biochemical approaches Approaches

Genetics

5-THF-homocysteine methyltransferase gene deletion construct being produced.

Heterologous expression

Delete gene of interest

methionine synthase prot., Met W No MeHg Met W + 3 genes in operon No MeHg 5-THF-homocysteine methyltransferase In progress

Transposon mutants

Reconstruct mutants and assay

Currently have ~1200 transposon mutants

ready for assay

Identify non-methylating mutants

Biochemistry

Protein Isolation

Identify Protein(s)

Create knock-out mutants and assay

Specific activity ~335 pmoles/mg/hr

[PmeI/EcoRV]

[EcoRV/SnaBI]

D. vulgaris Hildenborough (DvH) does not methylate mercury

Northern Blot

RT-PCR

Isolate RNA Electroporation

M (+) (-) 1 2 3 4 5 6

1 2 3 4 5 1 2 3 4 5 Spectinomycin aps A

M

75

500 300 200 75

700 1000 1500

bp

500 300 200

700 1000 1500

bp

GoI ND132(pRL27) colonies form within ~21 days on R:D plates

Conjugation

(KanR) Region of transposition (1710 bp)

Pick colonies into 96-well plates

Create a ~5000-10,000 member transposon library

Transposition


High-throughput MeHg+ assay

Analysis of Hg0 by CVAF or ICP/MS

“B” – “A” = MeHg+

Cell culture

HgCl2 spike

Hg2+

STEP 1 STEP 2

SnCl2 addition will reduce inorganic Hg, but not MeHg+, to Hg0

“A” “B”

Cell culture

Hg2+/ MeHg+

BrCl addition will oxidize MeHg+ to inorganic Hg, which is analyzed by SnCl2 reduction to Hg0


MD simulations revealed slow large-amplitude motions in Hg(II)-MerR related to an opening and closing of the DNA binding domains.

Apo-MerR

Hg(II)-MerR

20 Å

apo

Hg(II) RG = 24.7 ± 0.4 Å RG = 28.6 ± 0.5 Å ΔRG = 4 Å

Experimental SAXS data: A single Hg(II) ion binds to MerR causing a significant change in molecular structure.

• may be key mechanism for initiating mer operon transcription.

Guo et al., 2010. J. Molec. Biol., accepted

Once the methylation/demethylation genes are identified…


Direct protonation of the leaving group by Cys159

ΔE≠ = 35.6 kcal/mol

Cys159 is not coordinated With Hg at the TS

Proton transfer from Cys159 to Asp99

Asp99 protonates the leaving group

ΔE≠ = 22.4 kcal/mol Both Cys96 and Cys159 are coordinated with Hg.

Parks et al., 2009. J. Am. Chem. Soc. 131:13278.

Determined mechanism of MerB (organomercurical lyase; MeHg demethylase) function

Asp99

Cys96 Cys159

Mechanism I Asp99

Cys96 Cys159

Mechanism II


Systems model for Hg transformations at sediment-water interface

Critical gaps in scientific understanding:

 Oxidation, reduction, and species transformation

 Dominant chemical species and bioavailability

 Abiotic /biotic methylation and demethylation

 Biochemical pathways for methylation and demethylation

 Coupled biogeochemical reactions

 Surface catalyzed and photochemical reactions

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Acknowledgements

External Advisory Committee T Barkay (Microbiologist) R Wildung (Geochemistry) E Phillips (DOE-ORO) D Krabbenhoft (Hg geochemistry) J Blum (Hg geochemistry) *S Lindberg (Hg cycling) *R Mason (Hg geochemist)

ORNL Staff Liyuan Liang (Research Manager)

S Brooks (Field Investigations & Geochemistry) G Southworth A Biswas (post-doc) X Yen

B Gu (Fundamental Mechanisms) C Miller W Dong (post-doc) Y Bai (post-master)

D Elias (Microbial transformations) A Palumbo S Brown C Brandt J Moberly (post-doc) J Mosher (post-doc) T Vishnivetskaya (post-doc)

J Smith (Molecular simulations) A Johs J Parks H-B Guo

ORNL S&T Staff

BER ERSD Program Managers (Paul Bayer)

External Collaborators C Gilmour (Smithsonian) H Guo (UTK) K Kemner, B Mishra (ANL) S Miller (UCSF) K Nagy (UIC) J Schaefer, F Morel (Princeton) L Shi (PNNL) A Summers (UGA) J Wall, A Kucken (U Missouri) H Zhang (TN Tech)


Accomplishments to date

•  Significant scientific progress in all four tasks. –  Enhanced cross-task endeavours

•  6 papers submitted/in press/published. –  Additional 9 papers & 1 book partially supported by SFA.

•  12 manuscripts in preparation. •  19 conference presentations. •  New and renewed external collaborations. •  Scientific sessions: Goldschmidt, Meeting of the America’s, ACS, ASM

(being proposed). •  Numerous summer intern students.


Summary of FGA Design

The FGA was developed at ORNL and continues to be developed at ORNL and the University of Oklahoma. For a detailed description see: He, Z.L. et al. ISME J. 1, 67-77 (2007).

In 2010, the SFA has added new probes for merA and merB that will the total number of probes to >25,000.


Sequencing the Non-Methylating Transposon Mutants

Identified ND132 transposon mutants: 1-nucleoside ABC transporter, ATP-binding protein 2-chaperone protein DnaJ 3-Fe-S oxidoreductase 4-predicted proteasome-type protease 5-uncharacterized protein found in bacteria

Digest with NotI Ligate

Purify Plasposon

Uncut plasposon samples

Sequence out of transposon to

determine site of transposition

Isolate genomic DNA from each transposon mutant

NotI

NotI

Dennis, JJ and Zylstra, GJ (1998) Appl. Environ. Microbiol. 64: 2710-2715.

M 1 2 3 4 5

M

Transform E. coli

ND132(geneX::TnRL27)


Biochemical Analysis

Choi et al., 1994, Appl. Environ. Microbiol. 60: 1342-1346; 4072-4077.

Propyl iodide


Biochemical Analysis

Specific activity ~670 pmoles/mg cell protein after 20 hr

HgCl2 14CH3-THF

substrates added to

ND132 cells methylene

chloride

organic phase to scint. vial incubation

organic phase aqueous phase


Nested semi-random PCR2

PCR 1

Sequence out of transposon to

determine site of

transposition PCR2 product is sequenced with Primer 2

PCR 2

Primer 2

Primer 2 Primer 3

Primer 1 Semi-random primer mix

Semi-random primer mix (PCR1): CEKG2A: GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN AGA G CEKG2B: GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN ACG CC CEKG2C: GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN GAT AT CEKG2D: GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN AAC GC CEKG2E: GGC CAC GCG TCG ACT AGT ACN NNN NNN NNN TCG AC

2.Chun, KT et al. (1997) Yeast 13: 233-240.

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Progress in Understanding Hg Transformations: From ... · 1 Managed by UT-Battelle for the U.S. Department of Energy Progress in Understanding Hg Transformations: From Molecular Modeling