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2 Biochemistry and Genes Encoding Reductive Dehalogenases

2 Biochemistry and Genes Encoding Reductive Dehalogenases

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624



L. Adrian and F.E. Löffler



The possibility to heterologously express active reductive dehalogenases opens up

a wide field for new research. With more than 2000 putative reductive dehalogenase sequences identified, the majority without functional assignment, heterologous

expression is an important tool to characterize substrate specificity, substrate affinity, cofactor requirement, and to provide structural information. A high-throughput

expression pipeline is desirable to analyze the hundreds, or possibly thousands of

distinct reductive dehalogenases. Likely, the traditional approach to induce, enrich,

and purify reductive dehalogenases by chromatographic or electrophoretic techniques will still have value. Conventional biochemical approaches will also be

needed for the investigation of a recently identified larger respiratory complex in

Dehalococcoides mccartyi (Kublik et al. 2016), for which heterologous expression

and reconstitution may not be feasible.

With more structural information of reductive dehalogenases, comparative

analyses can reveal structure-function relationships, relevant binding motifs, and

contribute to predictive understanding of substrate range and reaction specificity.

Among the reductive dehalogenases, a system of orthologous clusters has been

established (Hug et al. 2013), and it will be a crucial task to refine this system as

new information becomes available. Also, the role of accessory proteins involved

in dehalogenase maturation should be studied (e.g., cofactor incorporation, folding,

transport across the cytoplasmic membrane, assembly of larger complexes). Most

promising are integrated approaches, and cross-disciplinary team research is most

likely to produce transformative discoveries that advance and broaden the field.

Although some progress has been made elucidating the controls of reductive

dehalogenase gene expression (Wagner et al. 2013; Kemp et al. 2013), the regulatory cascade is poorly understood. Regulatory genes are located in the vicinity of

reductive dehalogenase genes but virtually nothing is known about the inducing

molecules, how they interact with the transcription regulator(s), and how they trigger physiological responses. Also, it is unclear why different types of regulators

are involved in the transcriptional regulation of different reductive dehalogenase

genes. Understanding the regulation of reductive dehalogenation expression will

likely reveal a new conceptual understanding of regulatory circuits in prokaryotes.

Recently, it has been shown that reductive dehalogenases can display strong

substrate promiscuity and that relative reductive dechlorination rates follow electron density properties in the electron acceptor (Cooper et al. 2015). Apparently,

the traditional lock-and-key or induced-fit models for enzymes might not be

appropriate for reductive dehalogenases, and a more dynamic substrate binding

concept is needed. Further investigations are required using structural, kinetic,

and quantum chemistry information to analyze enzyme–substrate interplay and

electron transfer. This will also provide more detailed mechanistic insights into

reductive dehalogenation reactions, but more widely, will also contribute to better

understanding of coenzyme B12 dependent reactions.

Another research area of interest will be the elucidation of the components

and/or complexes involved in proton translocation across the membrane, and

if mechanistic differences distinguish phylogenetically distinct OHRB taxa.

Especially the electron transfer between the primary oxidizing protein complex



26  Outlook—The Next Frontiers for Research…



625



(e.g., a hydrogenase) and the reductive dehalogenase will need further attention

to identify electron carriers and their biochemical characteristics. These studies

will reveal if obligate OHRB (e.g., members of the Dehalococcoidia) and facultative OHRB (e.g., members of the Deltaproteobacteria) share electron transport

components or have distinct machineries to capture energy released in reductive

dechlorination reactions.

Reductive dehalogenases of OHRB share characteristic features including a

Tat leader peptide and require the so-called B protein with a putative membraneanchoring function. Genome analyses revealed that diverse taxa possess reductive dehalogenase genes not encoding these characteristic features. Experiments

with Comamonas sp. 7D-2 (Chen et al. 2013) and Nitratireductor pacificus strain

pht-3B (Payne et al. 2015) showed that such enzymes have reductive dehalogenase function albeit they are not directly involved in respiration. Similar reductive

dehalogenase genes were found in marine sediments. Possibly, these nonrespiratory reductive dehalogenases are part of degradation pathways that enable the host

to oxidize the chloroorganic compound and utilize an alternate electron acceptor

such as oxygen. Have organisms with nonrespiratory reductive dehalogenase lost

the ability to grow via organohalide respiration, or are they possibly the ancestors

that gave rise to the evolution of (obligate) OHRB?



26.3 Bioremediation Applications

Without extensive contamination of the environment with chlorinated chemicals

and ensuing public awareness and pressure, OHRB may not have been studied.

The reductive dechlorination process is a good example how practical needs

enable fundamental scientific discoveries while at the same time delivering solutions for pressing environmental problems. It is hoped that funding resources for

multidisciplinary team efforts will be available in the future to advance the science, generate economic opportunities, and elevate environmental cleanup from an

empirical practice to a science with predictable outcomes.

Bioaugmentation, the delivery of OHRB consortia into aquifers impacted with

chlorinated contaminants, can initiate or accelerate degradation and detoxification, as documented at many chlorinated solvent-contaminated sites (Ellis et al.

2000; Lendvay et al. 2003; Major et al. 2002; Löffler et al. 2013). D. mccartyi

appears to be crucial and only strains of this bacterial species have been demonstrated to detoxify chlorinated ethenes and produce environmentally benign

ethene. Interestingly, D. mccartyi strains are often present in contaminated aquifers but efficient ethene formation does not occur, presumably because the resident

Dehalococcoides populations lack the bvcA and/or vcrA reductive dehalogenase

genes required for vinyl chloride reductive dechlorination (Krajmalnik-Brown

et al. 2004; Müller et al. 2004). Thus, the complement of reductive dehalogenase

genes determines if the resident Dehalococcoides population efficiently degrades

the target contaminant(s). An interesting question is if bioaugmentation successes



626



L. Adrian and F.E. Löffler



really rely on the proliferation of the D. mccartyi strains introduced with the inoculum, or if the introduction of the genetic material encoding the vinyl chloride

reductive dehalogenase(s) is sufficient. Mounting evidence suggests that members

of the Dehalococcoidia acquire reductive dehalogenase genes via horizontal gene

transfer (McMurdie et al. 2011; Padilla-Crespo et al. 2014), which may offer alternative bioremediation strategies.

Detailed laboratory studies unravelled the complicated nutritional requirements of D. mccartyi strains. In addition to hydrogen, Dehalococcoides requires

other growth factors, foremost corrinoid, which is needed to assemble functional

reductive dehalogenases. The recent observation that the type or corrinoid (i.e.,

cobamides with different lower bases) has distinct effects on the reductive dechlorination performance of D. mccartyi strains expressing different vinyl chloride

reductive dehalogenases emphasizes the need to understand the roles of the community to support Dehalococcoides activity (Yan et al. 2015).

Metagenomics and metaproteomics enable a census of the genetic and actual

catalytic potential, respectively, of entire microbial communities. Such approaches

have not been effectively brought to bear at bioremediation sites but may be ideal

tools to develop systems understanding, which is needed to assess the complicated

interactions that govern activity of OHRB. Such detailed knowledge can inform

about reductive dechlorination potential, measure actual activity, and reveal interspecies dependencies, nutritional limitations, and possible synergistic effects, and

thus offer opportunities to refine bioremediation to efficiently achieve the desired

outcomes.



References

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Microbiol 89(6):1121–1139. doi:10.1111/mmi.12332

Cooper M, Wagner A, Wondrousch D, Sonntag F, Sonnabend A, Brehm M, Schüürmann G,

Adrian L (2015) Anaerobic microbial transformation of halogenated aromatics and fate

prediction using electron density modelling. Environ Sci Technol 49(10):6018–6028.

doi:10.1021/acs.est.5b00303

Ellis D, Lutz E, Odom J, Buchanan R, Bartlett C, Lee M, Harkness M, Deweerd K (2000)

Bioaugmentation for accelerated in situ anaerobic bioremediation. Environ Sci Technol

34(11):2254–2260. doi:10.1021/es990638e

Hug LA, Maphosa F, Leys D, Löffler FE, Smidt H, Edwards EA, Adrian L (2013) Overview

of organohalide-respiring bacteria and a proposal for a classification system for reductive

dehalogenases. Philos Trans R Soc Lond B Biol Sci 368(1616):20120322. doi:10.1098/r

stb.2012.0322

Kemp LR, Dunstan MS, Fisher K, Warwicker J, Leys D (2013) The transcriptional regulator CprK detects chlorination by combining direct and indirect readout mechanisms. Philos

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Krajmalnik-Brown R, Hölscher T, Thomson IN, Saunders FM, Ritalahti KM, Löffler FE (2004)

Genetic identification of a putative vinyl chloride reductase in Dehalococcoides sp. strain

BAV1. Appl Environ Microbiol 70(10):6347–6351. doi:10.1128/AEM.70.10.6347-6351.2004



26  Outlook—The Next Frontiers for Research…



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Kublik A, Deobald D, Hartwig S, Schiffmann C, Andrades A, von Bergen M, Sawers

RG, Adrian L (2016) Identification of a multiprotein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement. Environ Microbiol.

doi:10.1111/1462-2920.13200

Lendvay JM, Löffler FE, Dollhopf M, Aiello MR, Daniels G, Fathepure BZ, Gebhard M, Heine

R, Helton R, Shi J, Krajmalnik-Brown R, Major CL, Barcelona MJ, Petrovskis E, Hickey

R, Tiedje JM, Adriaens P (2003) Bioreactive barriers: a comparison of bioaugmentation and

biostimulation for chlorinated solvent remediation. Environ Sci Technol 37(7):1422–1431.

doi:10.1021/es025985u

Löffler FE, Ritalahti KM, Zinder SH (2013) Dehalococcoides and reductive dechlorination of

chlorinated solvents. In: Stroo HF, Leeson A, Ward CH (eds) Bioaugmentation for groundwater remediation, vol 5. SERDP ESTCP environmental remediation technology. Springer, New

York, pp 39–88. doi:10.1007/978-1-4614-4115-1_2

Major DW, McMaster ML, Cox EE, Edwards EA, Dworatzek SM, Hendrickson ER, Starr MG,

Payne JA, Buonamici LW (2002) Field demonstration of successful bioaugmentation to

achieve dechlorination of tetrachloroethene to ethene. Environ Sci Technol 36:5106–5116.

doi:10.1021/es0255711

McMurdie P, Hug L, Edwards E, Holmes S, Spormann A (2011) Site-specific mobilization of

vinyl chloride respiration islands by a mechanism common in Dehalococcoides. BMC

Genom 12(1):287. doi:10.1186/1471-2164-12-287

Müller JA, Rosner BM, von Abendroth G, Meshulam-Simon G, McCarty PL, Spormann

AM (2004) Molecular identification of the catabolic vinyl chloride reductase from

Dehalococcoides sp. strain VS and its environmental distribution. Appl Environ Microbiol

70(8):4880–4888. doi:10.1128/AEM.70.8.4880-4888.2004

Padilla-Crespo E, Yan J, Swift C, Wagner DD, Chourey K, Hettich RL, Ritalahti KM, Löffler

FE (2014) Identification and environmental distribution of dcpA, which encodes the reductive dehalogenase catalyzing the dichloroelimination of 1,2-dichloropropane to propene in

organohalide-respiring Chloroflexi. Appl Environ Microbiol 80(3):808–818. doi:10.1128/

AEM.02927-13

Payne KAP, Quezada CP, Fisher K, Dunstan MS, Collins FA, Sjuts H, Levy C, Hay S, Rigby

SEJ, Leys D (2015) Reductive dehalogenase structure suggests a mechanism for B12dependent dehalogenation. Nature 517(7535):513–516. doi:10.1038/nature13901

Wagner A, Segler L, Kleinsteuber S, Sawers G, Smidt H, Lechner U (2013) Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi. Philos Trans R Soc B

368:20120317. doi:10.1098/rstb.2012.0317

Yan J, Simsir B, Farmer AT, Bi M, Yang Y, Campagna SR, Löffler FE (2015) The corrinoid

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Index



A

Allyl chloride, 140

Ampicillin, 140

Anaerobic degradation, 52

Anaerobic wastewater treatment, 50

Anaeromyxobacter, 239

Anaeromyxobacter dehalogenans, 253

Apparent kinetic isotope effect (AKIE), 437,

598

Aroclor, 541, 542, 544–555, 550, 558–560,

563

Arsenate, 213, 218, 220

ATP generation, 45

B

Base-off conformation, 490

β-hexachlorocyclohexane, 155

Bioaugmentation, 5, 523, 528, 550, 557, 560,

563

Biochemistry, 623

Bioelectrochemical systems, 500

Biogeochemical cycling of organohalides, 328

Biokinetic models, 320

Bioremediation applications, 517, 625

Biostimulation, 143, 520, 563

B protein, 249

Bromide, 140

Brominated ethenes, 223

Bromophenols, 241

C

Carbon–Halogen bonds, 44

Carbon isotopes, 592

Catabolic transposon, 194

Catalase, 217



CblC, 490

cDCE, 222, 244–246

Cell envelope, 157

Chlordecone, 40

2-chlorophenol, 240, 243

Chlorinated solvent, 52

Chlorinated ethenes, 492, 517

Chlorinated methanes, 223

Chlorine isotopes, 594

Chlorobenzenes, 563

Chlorobenzoate, 236, 241, 243

Chloroethenes, 193

Chloroflexi, 143, 544, 545, 547, 550, 558, 559

3-chloro-4-hydroxyphenylacetic acid, 175

Chlorophenols, 194

Clostridia, 187

Cobalamin–halide complex, 493, 495

Cobalamin riboswitches, 199

Co-contaminants, 284

CO2 fixation, 164, 226

Co-metabolism, 31, 32

Commercial PCB, 541–544, 546, 551, 559

Commercial scale, 524

Commitment to catalysis, 598

Competition, 283, 284, 316

Complex I, 164, 224

Composite transposon, 161

Compound-specific isotope analysis (CSIA),

429, 587

Consumption threshold concentrations, 246

Corrinoid auxotrophy, 167

Corrinoid-binding domain, 489

Corrinoid biosynthesis, 199, 225, 228, 247

Corrinoid cofactor, 160, 248, 249, 345, 351,

352, 356

CprK, 346, 354, 366

CRISPR, 225



© Springer-Verlag Berlin Heidelberg 2016

L. Adrian and F.E. Löffler (eds.), Organohalide-Respiring Bacteria,

DOI 10.1007/978-3-662-49875-0



629



Index



630

Crystal structures, 487

Cytochromes, 217

D

Debromination, 196

Dehalobacter, 153, 213, 346, 348, 350–353,

356, 362, 364, 365, 548, 549, 557–559

Dehalobacter restrictus, 55

Dehalobium chlorocoercia, 550, 559, 563

Dehalococcoides, 213, 346, 347, 350, 353,

357, 360

Dehalococcoides mccartyi, 56, 518, 544–549,

551–553, 555–560

strain 195, 56, 549

strain CBDB1, 57, 551

strain FL2, 57

Dehalococcoidia, 143, 622

Dehalogenimonas, 137, 559

Dehalogenimonas alkenigignens, 138

Dehalogenimonas lykanthroporepellens, 137

Dehalospirillum, 210

Deiodination, 197

Deltaproteobacteria, 235, 250

Desulfitobacterium, 173, 213, 346, 348, 350,

352, 353, 356–359, 362, 364, 367

Desulfoluna spongiiphila, 241

Desulfomonile limimaris, 242

Desulfomonile tiedjei, 55, 228, 236, 253

Desulfovibrio dechloroacetivorans, 243

Desulfuromonas chloroethenica, 245

Desulfuromonas michiganensis, 245

Dibromoethane, 140

Dibromoethene, 223

Dibromophenols, 492

Dibromopropane, 140

Dichlorobenzene, 155

Dichloroethane, 137, 156, 244

Dichloromethane, 156

Dichlorophenol, 223

Dichloropropane, 137

Dihaloelimination, 33, 37, 140, 189

Dimethylbenzimidazole, 249

Dimethylsulfoxide, 139

DNAPL, 529

Dual-element isotope approach, 599

E

Ecology, 623

Ecophysiology, 248

Electron acceptors, 218, 220

Electron donor, 218, 518, 520

Electron shuttling, 247



Electron transfer, 494

Electrostimulation, 563

Energetics, 32

Enrichment (culture), 544–549, 552–554,

557–559

Environmental distribution of OHRB, 64, 81,

83, 88

Epsilonproteobacteria, 209

Equilibrium isotope effect (EIE), 589

Evolution, 378, 386

Extracellular electron transfer, 502

F

Facilitated fermentation, 261

Fatty acid composition, 180

Fermentation, 42, 213, 219

Fermentative growth, 183

Fermenters, 312, 315, 316, 317

Ferredoxin domain, 490

Ferredoxin-reductase, 491

Ferric iron, 237, 244, 246

4Fe-4S clusters, 160

Firmicutes, 187

Fluorobenzoate, 241

Food webs, 313

Fractured bedrock, 529

Fumarate, 237, 244, 245

Functional heterologous expression of RDase, 200

Fungi, 248

G

Gene cluster, 161

Genome, 225

Genome analyses, 250

Geobacter lovleyi, 246, 253

Geobacter sulfurreducens, 248

Geobacter thiogenes, 247

Gibbs free energy, 34, 39

Groundwater, 214

Growth factor requirements, 158

Growth yield, 5, 162, 240, 245

H

Habitat, 157

Halogen ligation, 493

Heme, 249

Homoacetogens, 314, 315, 317, 321

Horizontal gene transfer, 250

Hydrogen, 139, 213

Hydrogenase genes, 224

Hydrogenases, 163



Index

Hydrogen isotopes, 592

Hydrogenolysis, 33, 189

Hydrogen thresholds, 39

Hydrothermal vent, 251

I

Inhibition, 283, 284

Interspecies H2 transfer, 89, 309, 315

Intraprotein electron transfer, 492

Intrinsic kinetic isotope effect, 598

In situ bioremediation, 519

Iodophenols, 241

Iron–sulfur cluster, 491

Iscu, 228

Isotope enrichment factor, 435

Isotope fractionation, 589

Isotope ratio, 588

Isotope ratio mass-spectrometers (IRMS),

432, 592

Isotopic fractionation, 567

K

Kinetic isotope effect (KIE), 435, 589

Kinetic model, 286

Kinetics, 563

L

Low permeability zones, 534

Low pH aquifers, 530

M

Manganate, 218, 220

Marine and estuarine environments, 84

MarR regulators, 365, 366

Mathematical models, 534

Maturation, 227

Mechanism, 495

Membrane-bound hydrogenase, 224

Menaquinone, 157, 199, 217

Mesophilic, 139

Methanogens, 312, 314–317

Microaerophilic, 217

Mixotrophy, 160

Mobile element, 194

Myxococcales, 239

N

Natural organohalogen compounds, 7

Nitrate, 217, 218, 220, 223, 237, 242



631

Nitrate reductase, 224

Nitrite reductase, 220

Nitrogenase, 224

Nitroreductase fold, 489

Norpseudovitamin-B12, 495

NpRdhA, 489

O

O-demethylation, 183

OHRB generalists, 622

OHRB specialists, 622

OHR region, 227

Organohalide-respiring bacteria, 63, 64, 284

Organohalide respiration, 4, 33, 50, 55, 64,

345, 355, 363, 365, 368, 378, 384–386,

389, 549, 550

Oxidase, 217

Oxidation-reduction potential, 138

Oxidative transformations, 54

Oxygen, 217, 319

P

PCB dechlorination, 542–545, 547–549, 552,

555, 557–560

PCB reductive dehalogenase, 541, 556, 560,

557–560

PCE, 244–246, 541, 542, 548, 549, 553,

555–558, 560

PceA, 160, 213, 489

(Per)fluorinated compounds, 45

Peptidoglycan type, 157

Peptococcaceae, 187

Peroxidase, 228

Pesticides, 51

Phenol, 241

Phylogenetic diversity, 64, 73

Polychlorinated biphenyl, 563

Primary isotope effect, 595

Pristine environments, 248

Propenes, 223

Proton transfer, 494

Pyruvate, 178, 237, 241–243

Q

Quinol dehydrogenase, 228

R

Rate limitation, 442

Rayleigh equation, 591

RdhA active site, 492



Index



632

RdhA maturation, 491

RdhC, 228

Rebound, 532

Redox mediator, 503

Reductive acetyl-CoA pathway, 199

Reductive debromination, 244

Reductive dehalogenase (RDase), 249, 250, 287,

345, 346, 350, 353, 355–357, 360–362,

366, 377–383, 383–389, 541, 542, 548,

549, 551, 552, 555–558, 560, 623

Regulation, 227

Regulatory issues, 531

Remediation technologies, 283, 518

Respiratory processes, 4

Respiratory reductive dehalogenation, 4

Riboswitches, 167

S

Secondary isotope effect, 595

Sediment, 251

Sediment-free culture, 546, 547, 553, 559

Selenate, 218, 220

Sludge, 252

Solid-state electrodes, 499

Sponge, 251

Stable isotope fractionation, 435

Stable isotope, 429

Structure–function studies, 495

Substrate access channels, 492

Substrate specificity, 381

Sulfate, 237, 242

Sulfide, 138, 218

Sulfite, 220, 243

Sulfur, 217, 220, 247

Sulfurospirillum, 209, 211, 349, 351–353, 356,

357, 365

Sulfurospirillum carboxydovorans, 211, 214, 222

Sulfurospirillum deleyianum, 210

Sulfurospirillum halorespirans, 211, 213–215,

222, 227

Sulfurospirillum multivorans, 210, 213, 214,

222, 249

Sulfurospirillum species SL2, 209, 211, 349,

351–353, 356, 357, 365



Sulfurospirillum tacomaensis, 211, 222

Syntrophic relationship, 160, 213

T

TCE, 222, 244, 245

Tetrachloroethane, 137, 154, 244

Tetrachlorophthalide, 156

Tetrathionate, 220

Thermodynamics, 32, 34

Thiosulfate, 242, 243

Threshold concentrations, 246

Titanium-citrate, 138

Toxicity, 253

Transcriptional analysis, 555

Transcriptional regulator, 195

Transposon mutagenesis, 200

Tribromoethene, 223

Tribromophenol, 243

Trichlorobacter thiogenes, 247

Trichloroethane, 137, 155, 244

Trichloropropane, 137, 140

Twin-arginine motif, 160, 228

Two-component regulatory system, 228

Two-phase system, 154

Tyr-Lys/Arg motif, 492

U

Up-flow anaerobic-sludge bed reactor, 197

Uranium, 246

V

Vancomycin, 140

Vancomycin resistance gene cluster, 178

W

Wastewater reclamation, 52

Wood-Ljungdahl pathway, 164

Y

Yield, 240



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