Biogeochemical Controls Over Organohalide-Respiring Chloroflexi - - PowerPoint PPT Presentation

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Biogeochemical Controls Over Organohalide-Respiring Chloroflexi Frank Löffler

Center for Environmental Biotechnology Department of Microbiology Department of Civil & Environmental Engineering Bioscience Division, Oak Ridge Na@onal Laboratory

EPA, 2013

Contaminated Sites in the U.S.

1,322 Superfund sites 3,747 RCRA sites >450,000 Brownfields

166 MILLIO N

PEO

PLE

• f a Superfund or a

Live

3

MILES

RCRA Correc@ve

53%

OF THE U.S.

PO PULATIO N

Including Within Ac@on Site, Equal to

55% OF ALL

CHILDREN

UNDER AGE 5

... and in Europe

Majority of sites impacted with chlorinated compounds

2

Reductive Dechlorination: A Process that Leads to Contaminant Detoxification In Situ

2[H] 2[H] 2[H] 2[H] HCl HCl HCl HCl Energy Energy Energy Energy

Organohalide Respiration

Freedman, D. L., and J. M. Gossett. 1989. Appl. Environ. Microbiol. 55:2144-2151 He et al. 2003. Nature. 424:62-65

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Populations Involved in Reductive Dechlorination of CEs

2[H] 2[H] 2[H] 2[H] HCl HCl HCl HCl

Dehalococcoides mccartyi

Geobacter lovleyi, Dehalobacter, Sulfurospirillum, Desulfuromonas, Desulfitobacterium

Sung et al. 2006 AEM, 72:2775 Löffler et al. 2013 IJSEM, 63:625 4

PopulaMons Involved in ReducMve DechlorinaMon

• f Chlorinated Ethenes

1,173 GW samples, 111 sites 849 samples: Dhc & Dhgm 65%: Dhgm outnumber Dhc

Yang et al. ISME J. 2017. 11:2767-2780

He et al. 2003, Nature, 424:62

5

Populations Involved in Reductive Dechlorination

He et al. 2003, Nature, 424:62 Löffler et al. 2013, IJSEM, 63:625

• f Chlorinated Ethenes

1,173 GW samples, 111 sites 849 samples: Dhc & Dhgm

Dehalococcoides (Dhc)

65%: Dhgm outnumber Dhc

Dehalogenimonas (Dhgm)

Yang et al. ISME J. 2017. 11:2767-2780

He et al. 2003, Nature, 424:62

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Structural Basis of Organohalide RespiraMon

Dimer of PceA

Bommer et al. 2014. Science, 346:455 Payne et al. 2015. Nature, 517:513

Corrinoid Cofactor 2 FeS Clusters

Sulfurospirillum mul0vorans

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Corrin ring Upper ligand Cyanocobalamin Vitamin B12 Methylcobalamin Adenosylcobalamin

5’deoxyadenosyl Methyl Cyano

Lower base Dimethylbenzimidazole (DMB)

8

Lower Bases

Benzimidazole (Bza) type

OH OCH3 OCH3 N N N

Corrin ring

N N N CH3 CH3 CH3 N N N N N CH3 N N N N N NH2 CH3 N N N N NH2 SCH3 N N N N NH2 SCH3 O N N N N NH2 SCH3 O O N N N N OH NH N N N O NH2

Nucleobase type

N N N NH2

Purine

N

Phenol type Naphthimidazole

Yan et al. 2018.

• Nat. Chem. Biol.

14:8-14.

N O O CH3 N

9

De novo Biosynthesis of Corrinoids

Bacteria Archaea

(n = 56,902) (n = 1,362)

Geobacter lovleyi

• Corrinoid prototroph

Dehalococcoides mccartyi

Complete Incomplete

• r missing
• Corrinoid auxotroph

Incomplete

• r missing

Complete

10

Dhc & Dhgm: Strict Requirement for Corrinoid

cDCE VC ETH

No B12 Ethene Dhc strain BAV1 Sufficient B12 [25 µg/L] cDCE VC Limited B12 [1 µg/L] No B12 No dechlorination VC stall Complete Dechlorination (Detoxification)

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No B12

Specific Aims

• Aim 1: Explore the specific cobamide requirements of organohalide-

respiring Dhc relevant for detoxifica@on of chlorinated ethenes

• Aim 2: Demonstrate that geochemical condi@ons affect the specific

cobamide pool, and hence Dhc ac@vity

• Aim 3: Iden@fy community and Dhc biomarkers that indicate when

cobamide and/or lower base bioavailability limit Dhc reduc@ve dechlorina@on ac@vity

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Simplified Model

H2

• f Organohalide

Respiration

Dehalococcoides mccartyi strain BAV1

2 e-

Dehalococcoides mccartyi

• Corrinoid auxotroph

ATP

Geobacter lovleyi

• Corrinoid prototroph

H+

2 H+ H+

R-Cl + H+

Corrinoid' Cofactor'

R-H + Cl-

13

Who Supplies Corrinoid to Dhc and Dhgm?

Geobacter lovleyi

Dehalococcoides mccartyi

Corrinoid +

?

Corrinoid –

14

Co-Culture Experiments: Corrinoid Producer / Dhc mccartyi

Cobamide producing microbe Dhc mccartyi

Corrinoid auxotroph

Geobacter spp. Strain BAV1 Strain FL2

?

Dhc dechlorination activity? 15

Co-Cultures Dhc Growth Corrinoid Producer Dhc Strains Geobacter lovleyi BAV1, FL2 +

Summary of Co-Culture Experiments

Dhc mccartyi Cobamide producing microbe Dhc dechlorination activity?

?

Strain BAV1 Strain FL2 Geobacter spp.

Corrinoid auxotroph

Geobacter sulfurreducens BAV1, FL2

• Sporomusa sp.

BAV1, FL2, GT

• Acetobacterium sp.

BAV1, FL2

• Clostridium aceticum

BAV1, FL2

• Methanosarcina barkeri

BAV1, FL2, GT

• 16

Summary of Co-Culture Experiments

Co-Cultures Corrinoid Producer Geobacter lovleyi Geobacter sulfurreducens Sporomusa sp. Acetobacterium sp. Clostridium aceticum Methanosarcina barkeri Dhc Growth Dhc Strains BAV1, FL2 + BAV1, FL2

• BAV1, FL2, GT
• BAV1, FL2
• BAV1, FL2
• BAV1, FL2, GT
• Dhc

mccartyi Cobamide producing microbe Dhc dechlorination activity?

?

Strain BAV1 Strain FL2 Geobacter spp.

Corrinoid auxotroph

Dhc Growth with DMB + + + + + +

Yan et al. 2012. Appl. Environ. Microbiol. 78:6630-6636 Yan et al. 2013. Phil. Trans. R. Soc. B. 368, 20120320

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Co-Culture Experiments

Co-Cultures

Dhc mccartyi Cobamide producing microbe Dhc dechlorination activity?

?

Strain BAV1 Strain FL2 Geobacter spp.

Corrinoid auxotroph

Acetobacterium sp. BAV1, FL2

• Clostridium aceticum

BAV1, FL2

• Methanosarcina barkeri

BAV1, FL2, GT

• Yan et al. 2012. Appl. Environ. Microbiol. 78:6630 6636 Yan et al. 2013. Phil. Trans. R. Soc. B. 368, 20120320

Corrinoid Producer Geobacter lovleyi Geobacter sulfurreducens Wildtype Geobacter sulfurreducens + pNJ052 Dhc Growth Dhc Strains BAV1, FL2 + BAV1, FL2

• BAV1, FL2

+

• 18

/ t eA eA cA rA

Key Dhc RDases

Dhc str. 195 pceA p eA PceA/PteA tc TceA Dhc str. FL2 tc TceA Dhc str. BAV1 bv BvcA Dhc str. VS Dhc str. GT vc VcrA

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Lower Base Affects Dechlorination Activity

[DMB]Cba

Dechlorination rates [mmoles Cl- L-1 d-1]

Strain BAV1 (BvcA)

[Ben]Cba cDCE, VC and ethene [µmoles/bottle]

Time (days)

Yan et al. 2016. ISME J. 10:1092–1101

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Lower Base Affects Dechlorination Activity

Strain GT (VcrA)

Dechlorination rates [mmoles Cl- L-1 d-1]

cDCE, VC and ethene [µmoles/bottle] [DMB]Cba [Ben]Cba Time (days)

Lower base affects dechlorination rates and endpoints

Yan et al. 2016. ISME J. 10:1092–1101

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Nitr Su Me Ir l Ferme

• n

f a at t te h

• e

Re a

• Re

Re n d n

• u

d d t g i c u u e n i n c c n g i i i g n n c g g

Corrinoid Production Under Different Redox Conditions

Third Creek Site Knoxville, TN

• Metal-manufacturing
• Chlorinated solvents

Sediment

MW 7

GW

• Commerce Street Superfund Site

Williston, Vermont

• Mul@-tenant industrial park
• TCE, cis-DCE, petroleum

hydrocarbons, metals (chromium, cadmium, nickel)

Corrinoid Extraction and Identification

22

Corrinoids Produced by the Community Under Different Redox Conditions

Corrinoids(( produced( fermen2ng( Methanogenic( Iron(( reducing( Sulfate( reducing( Nitrate( reducing(

+

• +
• +

+ + + + + + +

• Glucose

Lactate(

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• 5-Hyd

Dimet

Redox Conditions Affect Corrinoid Type(s) and Quantity

OH N

5-OHBza-Cba

N

• Glucose

fermenting CH4 Nitrate reducing Sulfate Iron

ng

CH3 N

MeBza-Cba

N CH3 N

DMB-Cba

N CH3 O

Phe-Cba

O CH3 Cre-Cba

reducing reducing

Seus et al. 2019. In Preparation

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Corrinoid Quantity and Quality Determine Dhc Activity

No B12 Limited B12 [1 µg/L] Sufficient B12 [25 µg/L]

cDCE VC ETH

25

NaBH4 N2O

Co(III) Co(II) Co(I)

Banks et al. 1968. J. Chem. Soc. A, 2886

Impact of N2O on corrinoid-dependent reductive dechlorination?

26

Dechlorination (% Cl- released)

10 20 60

N2O Inhibits Corrinoid-Dependent Reductive Dechlorination

Geobacter lovleyi

N2O [µM]

Geobacter lovleyi

N2O [µM]

(PCE) (Fumarate)

PCE TCE cDCE

PCE, TCE, cDCE (µmol/bottle)

Fumarate, succinate (mM) 20 100

Time (days) Time (days) 27

Impact of Increased N2O in Environmental Systems

N2O (µM) 0.3 12.5 37.4 75 84 143 65.7

Ki, N2O = 40.8 ± 3.8 µM Ki, N2O = 21.2 ± 3.5 µM Ki, N2O = 9.6 ± 0.4 µM

Yongchao Yin

Yin et al. 2019. Environ. Sci. Technol. Nitrous Oxide is a Potent Inhibitor of Bacterial Reductive Dechlorination. 53:692-701 Jurado et al. 2017, Sci. Total Environ. 584–585:207-218

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cbiB cbiP btuR cobU cobS cobD cobC cobT

btuF-btuC-btuD ABC transport

btuR cbiZ cobU Uroporphyrinogen III Precorrin-2

Co++ Co++

cbiK

cbiM-cbiN-cbiQ-

Adenosylcobalamin

cysG cbiX cobI

cbiO ABC transport

Precorrin-3A

O2

Cobalt-precorrin-2

Biosynthesis

cobG cbiL Cobalt-precorrin-3

Early cobalt

Precorrin-3B cobJ cbiH

inserMon

Cobalt-precorrin-3 Precorrin-4 cbiF

pathway

cobM Cobalt-precorrin-4 Precorrin-5 cbiG cobF Cobalt-precorrin-5 Precorrin-6A

Late cobalt

cbiD cobK Cobalt-precorrin-6A

insertion

cbiJ Precorrin-6B

pathway

cobL Cobalt-precorrin-6B

Corrinoid

Precorrin-8x cbiE

Salvage

cobH Cobalt-precorrin-7 cbiT Hydrogenobyrinic acid Cobalt-precorrin-8x

btuF-btuC-btuD

cobB cbiC

ABC transport

Hydrogenobyrinic acid a,c-diamide Co++ Cobyrinic acid

Archaeal Salvage

Incomplete corrinoid cobN cbiA Cob(II)yrinic acid a,c-diamide Ado- ? cobinamide btuR Cob(I)yrinic acid a,c-diamide cbiZ

Dehalococcoides

cbiP Ado-Cobyric Ado- Ado-cob(I)yrinic acid a,c-diamide

genes

btuR acid cobinamide Ado-cobyric acid cobU Ado- L-threonine- 1-amino-2-propanol- cobinamide- phosphate cbiB phosphate phosphate cobD Ado-cobinamide- phosphate

Bacterial Salvage

cobU Ado-GDP-cobinamide 5,6-dimethylbenzimidazole alpha-ribazole- alpha- 5-phosphate Ribazole cobT cobC cobS

Adenosylcobalamin

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High-Throughput qPCR

• Monitor many biomarker genes

simultaneously

QuantStudio Open Array Plate 3.072 through-holes 48 subarrays x 64 through-holes/subarray

Hydrophilic Hydrophobic 33 nL through-holes

• Scalable platform (224x12; 112x24; 56x48)
• Cost per reaction reduced from $3.0 to

$0.30

• Four plates can be cycled simultaneously,

producing up to 12,288 qPCR data points per run Robot reduces pipetting errors

Kara Murdoch et al. 2019. In Preparation

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Take Home Messages

• Corrinoids are essential for most organisms
• Corrinoid-auxotrophic OHRBs (e.g., Dhc, Dhgm) are ideal systems

to study corrinoid effects on metabolism

• Corrinoid quantity (flux) affects dechlorination activity
• Corrinoid type (lower base) determines reductive dechlorination rates

and end points (i.e., function) in Dhc

• Geochemistry affects corrinoid pool
• Purine is a naturally occurring lower base
• New avenues to manipulate microbal metabolism

(function, ecology • biotechnology, medicine)

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Peer-reviewed Manuscripts

Yin, Y., Yan, G. Chen, F. Kara Murdoch, N. Pfisterer, and F.E. Löffler. 2019. Nitrous oxide is a potent inhibitor of bacterial reduc@ve dechlorina@on. Environ. Sci. Technol. 53:692-701 | doi: 10.1021/acs.est.8b05871 Yan, J., M. Bi, A.K. Bourdon, A.T. Farmer, P.-H. Wang, O. Molenda, A. Quaile, N. Jiang, Y. Yang, Y. Yin, B. Şimşir, S.R. Campagna, E.A. Edwards, and F.E. Löffler. 2018. Purinyl-cobamide is a native prosthetic group of reductive dehalogenases. Nat. Chem. Biol. 14:8-14. | doi:10.1038/nchembio.2512 Clark, K., D.M. Taggart, B.R. Baldwin, K.M. Ritalahti, R.W. Murdoch, J.K. Hatt, and F.E. Löffler. 2018. Normalized quantitative PCR measurements as predictors for ethene formation at sites impacted with chlorinated ethenes. Environ. Sci. Technol. 52:13410-13420 | doi: 10.1021/acs.est.8b04373 Yang, Y., S.A. Higgins, J. Yan, B. Şimşir, K. Chourey, R. Iyer, R.L. Hesch, B. Baldwin, D.M. Ogles, and F.E. Löffler. 2017. Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reduc@ve dehalogenase genes. The ISME Journal. 11:2767-2780. | doi: 10.1038/ismej.2017.127 Yan, J., B. Şimşir, A.T. Farmer, M. Bi, Y. Yang, S.R. Campagna, and F.E. Löffler. 2016. The corrinoid cofactor of reduc@ve dehalogenases affects dechlorina@on rates and extents in organohalide-respiring Dehalococcoides mccartyi. ISME J. 10:1092-1101. | doi: 10.1038/ismej.2015.197 32

Team Corrinoid

• Dr. Fadime Kara Murdoch
• Dr. Jun Yan

Steven Higgins Yongchao Yin Laurel Seus Meng Bi Nannan Jiang Burcu Şimşir Amanda Devolk Yi Yang UTK Chemistry Microbial Insights, Inc. Abigail Farmer, Allen Bourdon, Dora Ogles-Taggart, Kate Clark, Brep Baldwin Alex Fisch, Shawn Campagna University of Toronto Po-Hsiang (Tommy) Wang, Elizabeth Edwards

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