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8 Functional Diversity of Reductive Dehalogenases in the Dehalobacter Genus

8 Functional Diversity of Reductive Dehalogenases in the Dehalobacter Genus

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J. Maillard and C. Holliger

Fig. 8.3  Protein sequence likelihood tree analysis of all putative reductive dehalogenases identified in Dehalobacter spp. Each sequence is given by its gene index (GI) reference number and

an abbreviation for the species and strain. A colour code is used to distinguish the strains: red

Dehalobacter restrictus; orange Dehalobacter sp. strain E1; yellow Dehalobacter sp. strain

UNSWDHB; light blue Dehalobacter sp. strain CF; dark blue Dehalobacter sp. strain DCA;

green Dehalobacter sp. strain FTH1; grey Dehalobacter sp. in co-culture or enrichment cultures.

Legend: Dre: Dehalobacter restrictus; Dhb: Dehalobacter sp.; Dde: Desulfitobacterium dehalogenans; Ddi: Desulfitobacterium dichloroeliminans; Dha: Desulfitobacterium hafniense; Dmc:

Dehalococcoides mccartyi; Smu: Sulfurospirillum multivorans

D. dehalogenans (left side of the tree), and another class which contains many

diverse RdhA sequences (right side of the tree). This distinction is further validated when considering the genetic structure of rdh operons in D. restrictus

8  The Genus Dehalobacter


(Rupakula et al. 2013). Indeed, a variety of gene clusters was identified there, with

the cprA-like genes being embedded in a simple rdhBA operon structure, while the

other ones are part of rdhBAC, rdhABC or rdhABCT structures. This suggests that

the numerous cprA-like genes in Dehalobacter are likely the result of relatively

recent gene duplications.

As for other RdhA in general (see (Hug et al. 2013) and Chap. 16), the

sequence-substrate relationships in Dehalobacter RdhAs is impossible to establish. Too scarce knowledge is available not allowing any clear correlation between

sequence features and substrate specificity. As an example, in the group of the best

defined and closely related RdhA enzymes in Dehalobacter (indicated by a double

asterisk in Fig. 8.3), two substrates were identified, PCE (and TCE) and 1,2-DCA.

8.9 Corrinoid Metabolism in Dehalobacter spp.

As already stated above, D. restrictus has been characterized as a corrinoid

auxotrophic bacterium (Holliger et al. 1998). The elucidation of the genome

sequence of D. restrictus (Kruse et al. 2013), but also other Dehalobacter spp.

(Deshpande et al. 2013; Maphosa et al. 2012; Tang et al. 2012) gave new insights

in the corrinoid metabolism and more specifically in the corrinoid biosynthesis pathway. At the very first look it was surprising to identify the complete

corrinoid biosynthetic pathway in D. restrictus. However, a deletion mutation affecting the cbiH gene was then proposed to be responsible for its corrinoid auxotrophy (Kruse et al. 2013; Rupakula et al. 2013). In a proteomic study,

several proteins of this pathway, including CbiH, were not detected in the proteome of D. restrictus cultivated in standard growth conditions (i.e. in presence

of 250 μg/L of cobalamin in the medium) (Rupakula et al. 2013). A functional

genomic study was then conducted to investigate the effect of corrinoid limitation on corrinoid metabolism (Rupakula et al. 2015). Five distinct operons were

characterized in D. restrictus and two major differences were observed in the

genomes of other Dehalobacter spp., namely the presence of an intact copy of

cbiH and the lack of operon-2, which encodes several corrinoid transport proteins and proteins involved in corrinoid salvaging. All five operons are regulated by cobalamin riboswitches (Rupakula et al. 2015), baring similarity to D.

hafniense (Choudhary et al. 2013). The comparison of the proteome from D.

restrictus cells cultivated in the presence of 250, 50 and 10 μg/L of cobalamin

in the medium revealed a strong up-regulation of proteins encoded in operon-2,

suggesting that D. restrictus exploits an enhanced capacity of corrinoid transport and salvaging under corrinoid limitation. Sequence analysis further indicated that operon-2 of D. restrictus shows strong homology to an operon present

in Acetobacterium woodii, suggesting that horizontal gene transfer may have

occurred (Rupakula et al. 2015). So far, scarce information is available on corrinoid metabolism at the physiological level in other Dehalobacter strains. It was

suggested that Dehalobacter sp. strain E1 might benefit from corrinoids provided


J. Maillard and C. Holliger

by Sedimentibacter sp. present in the co-culture (Maphosa et al. 2012). When

indicated, cultures of Dehalobacter spp. were always cultivated in the presence

of cobalamin. Hence, further work is needed to test the ability of Dehalobacter

strains that have an intact cbiH gene to synthesize corrinoids de novo.

8.10 Concluding Remarks

Dehalobacter in pure culture or enrichments seem to be dedicated to organohalide

degradation, either by reduction in organohalide respiration or by fermentation as in

the case of DCM. For the fermentative strains it is however not known whether they

have additional metabolic capabilities not involving an organohalide. The recently

available genome sequences and the recently isolated new strains provide new avenues of research for this bacterial genus. The increasing number of scientific publications dealing with Dehalobacter populations since 2009, about 70 in total, indicates

that we will learn a lot more about the Dehalobacter genus in the near future.


Choudhary PK, Duret A, Rohrbach-Brandt E, Holliger C, Sigel RKO, Maillard J (2013)

Diversity of cobalamin riboswitches in the corrinoid-producing organohalide respirer

Desulfitobacterium hafniense. J Bacteriol 195(22):5186–5195. doi:10.1128/jb.00730-13

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Dehalobacter UNSWDHB, a chloroform-dechlorinating bacterium. Genome Announc 1 (5).


Duret A, Holliger C, Maillard J (2012) The physiological opportunism of Desulfitobacterium

hafniense strain TCE1 towards organohalide respiration with tetrachloroethene. Appl Environ

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Appl Environ Microbiol 75(9):2684–2693. doi:10.1128/aem.02037-08

8  The Genus Dehalobacter


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Huntemann M, Wei CL, Han J, Chen A, Kyrpides N, Szeto E, Markowitz V, Ivanova

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sequence of Dehalobacter restrictus PER-K23(T.). Stand Genomic Sci 8 (3):375–388.


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Lee M, Low A, Zemb O, Koenig J, Michaelsen A, Manefield M (2012) Complete chloroform dechlorination by organochlorine respiration and fermentation. Environ Microbiol

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Environ Sci Technol 47(3):1534–1541. doi:10.1021/es303784f

Li Z, Suzuki D, Zhang C, Yoshida N, Yang S, Katayama A (2013b) Involvement of Dehalobacter

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Lima G, Parker B, Meyer J (2012) Dechlorinating microorganisms in a sedimentary rock matrix

contaminated with a mixture of VOCs. Environ Sci Technol 46(11):5756–5763. doi:10.1021/


Löffler FE, Yan J, Ritalahti KM, Adrian L, Edwards EA, Konstantinidis KT, Muller JA,

Fullerton H, Zinder SH, Spormann AM (2013) Dehalococcoides mccartyi gen. nov., sp.

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Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum

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Lowe M, Madsen EL, Schindler K, Smith C, Emrich S, Robb F, Halden RU (2002)

Geochemistry and microbial diversity of a trichloroethene-contaminated Superfund site

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Chapter 9

The Genus Desulfitobacterium

Taiki Futagami and Kensuke Furukawa

Abstract To date, 22 Desulfitobacterium strains have been isolated. From them

a total of six distinct species have been proposed: D. hafniense, D. dehalogenans,

D. chlororespirans, D. metallireducens, D. dichloroeliminans, and D. aromaticivorans. The isolated strains are strictly anaerobic, mesophilic, and grow in the

neutral pH range. The cells are slightly curved rods ranging from 2 to 7 μm in

length and 0.3 to 1 μm in width. Most of the Desulfitobacterium strains have

been isolated as organohalide-respiring bacteria (OHRB) and show versatile

dehalogenation of both chlorinated aliphatic and aromatic compounds such as

chloroethenes and chlorophenols. The Desulfitobacterium strains are phylogenetically classified into the phylum Firmicutes (Gram-positive bacteria). The closest related OHRB genus of Desulfitobacterium is Dehalobacter, the members of

which are strict OHRB within the phylum Firmicutes (see Chap. 8). In contrast,

the Desulfitobacterium strains isolated to date are not strict OHRB. In addition to

the ability to respire with organohalides, most isolates can grow fermentatively on

pyruvate and can utilize a variety of electron acceptors, including sulfite, thiosulfate, fumarate, Fe(III), and Mn(IV). Complete genome information is available for

four Desulfitobacterium strains and draft information is available for five strains.

The complete genomes range from 3.62 to 5.73 Mbp, with GC content ranging

from 44.2 to 47.5 % and the number of predicted coding sequences ranging from

3340 to 5060. Consistent with their physiological diversity, the Desulfitobacterium

genome has been shown to encode a variety of respiratory reductases, including

reductive dehalogenases.

T. Futagami (*) 

Education and Research Center for Fermentation Studies, Faculty of Agriculture,

Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan

e-mail: futagami@chem.agri.kagoshima-u.ac.jp

K. Furukawa 

Faculty of Food Science and Nutrition, Department of Food and Bioscience,

Beppu University, Kitaishigaki 82, Beppu, Oita 874-8501, Japan

e-mail: kfurukaw@nm.beppu-u.ac.jp

© Springer-Verlag Berlin Heidelberg 2016

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

DOI 10.1007/978-3-662-49875-0_9



Abbreviations Used in Text


3-Cl-4-OHPA3-chloro-4-hydroxyphenylacetic acid



DMSODimethyl sulfoxide

OHRBOrganohalide-respiring bacteria



RDaseReductive dehalogenase



Abbreviations Used in Tables




CDCarbon dichloride


3-Cl-4-OHPA3-chloro-4-hydroxyphenylacetic acid


CTCarbon tetrachloride

















VCVinyl chloride

T. Futagami and K. Furukawa

9  The Genus Desulfitobacterium


9.1 Discovery

The Desulfitobacterium strains have been isolated primarily as organohaliderespiring bacteria (OHRB) from organohalide-contaminated environments. The

first Desulfitobacterium isolate was strain DCB-2 (for [aryl-] dechlorinating bacterium no. 2, currently known as Desulfitobacterium hafniense DCB-2) (Madsen

and Licht 1992). Strain DCB-2 was isolated as a chlorinated phenol-degrading bacterium from a stable trichlorophenol (TCP)-dechlorinating consortium

enriched from municipal digestor sludge in Copenhagen, Denmark. At that time,

strain DCB-2 was reported to dehalogenate chlorophenols at the ortho and meta

positions. This activity differed from that reported for Desulfomonile tiedjei DCB1, which was the first isolate of the OHRB, dechlorinating only from the meta

position (Shelton and Tiedje 1984; DeWeerd et al. 1990). Strain DCB-2 was later

designated D. hafniense DCB-2 (Christiansen and Ahring 1996a). The species

name hafniense reflects that Copenhagen was the place of isolation.

The nomenclature of the genus Desulfitobacterium was defined upon isolation of the second strain, D. dehalogenans JW/IU-DC1, in Athens, Georgia, USA

(Utkin et al. 1994). The strain JW/IU-DC1 can dechlorinate at the ortho position

of both chlorophenols and chlorophenylacetates (Utkin et al. 1994, 1995) and was

the first Desulfitobacterium strain for which energy conservation through reductive

dechlorination of 3-chloro-4-hydroxyphenylacetic acid (3-Cl-4-OHPA) was shown

(Mackiewicz and Wiegel 1998). The genus name Desulfitobacterium describes a

rod-shaped bacterium that reduces sulfite. However, several Desulfitobacterium

isolates that do not reduce sulfite have been reported, including D. metallireducens

853-15A and Desulfitobacterium sp. PR (Finneran et al. 2002; Ding et al. 2014). A

total of 22 Desulfitobacterium strains exhibiting a wide dehalogenation spectrum

have been isolated and characterized to date.

9.2 Isolation and Habitat

Desulfitobacterium strains are distributed worldwide, having been isolated from

Denmark, the United States, Vietnam, The Netherlands, Germany, Canada, Japan,

Finland, and Poland (Table 9.1). Most Desulfitobacterium strains have been isolated during the course of studies of reductive dechlorination processes. Therefore,

the target organochlorines in each of these studies were generally used as the electron acceptors in enrichment cultures. A total of 15 Desulfitobacterium strains

(D. hafniense strains DCB-2, TCE1, TCP-A, PCE-S, PCP-1, Y51; D. dehalogenans JW/IU-DC1; D. chlororespirans Co23; D. dichloroeliminans DCA1; and

Desulfitobacterium. sp. strains Viet1, KBC1, PCE1, B31e3, JH1, PR) have been

isolated from enrichment cultures containing the organochlorines tetrachloroethene (PCE), trichloroethene (TCE), 2,4,6-TCP, 2,3-dichlorophenol (2,3-DCP),

pentachlorophenol (PCP), 3-Cl-4-OHPA, 1,2-dichloroethane (1,2-DCA), or 1,1,1trichloroethane (Suyama et al. 2001; Gerritse et al. 1999; Breitenstein et al. 2001;

T. Futagami and K. Furukawa


Table 9.1  Desulfitobacterium strains in order of the year they were first reported


Stock center no. Source of isolation



Municipal digestor

D. hafniense


A freshwater

D. dehalogenans JW/ DSM 9161,

ATCC 51507

sediment collected


from a pond of a

wooded area

DSM 10344

Soil contaminated


with chloroethene

sp. PCE1

ATCC 700175, Compost soil

D. chlororespirans

DSM 11544


D. hafniense PCP-1 ATCC 700357, A mixture of

DSM 12420

anaerobic sewage

sludge and soil

samples that had

been contaminated

with PCP

Soil contaminated

D. hafniense PCE-S DSM 14645

with chloroethenes



sp. Viet1

D. dehalogenans



D. hafniense TCE1

DSM 12704a

D. hafniense TCP-A

DSM 13557

D. hafniense DP7

DSM 13498

D. hafniense Y51

NBRC 109954

D. hafniense GBFH


D. metallireducens




ATCC 700041

Geographic origin



The Sandy Creek

Nature Park,

Athens, Georgia,


The Netherlands


Michigan, USA

Quebec, Canada



Parfume River


A freshwater

Huế, Vietnam

Soil obtained from

a chloroethenepolluted location

Sediment of the

river Saale

Fresh fecal sample

of a healthy

28-year-old female


Soil contaminated

with PCE



Breda, The



anaerobic aquifer


Athens, Georgia,



The Netherlands


Madsen and

Licht (1992)

Utkin et al.


Gerritse et al.

(1995, 1996)

Sanford et al.



et al. (1996)

Miller et al.

(1997), Goris

et al. (2015)

Löffler et al.


Wiegel et al.

(1999), ATCC


Gerritse et al.



et al. (2001)

van de Pas

et al. (2001b)

Fukuoka, Japan

Suyama et al.



Coeur d’Alene

River delta, Lake et al. (2001)

Coeur d’Alene,

Idaho, USA

Floodplain of the Finneran et al.


San Juan River,

Shiprock, NM,



9  The Genus Desulfitobacterium


Table 9.1  (continued)


Stock center no. Source of isolation


Subsurface smecD. hafniense G2

tite bedding of

the Twiggs Clay

formation of late

Eocene age

Soil matrix of an

D. dichloroeliminans BCCM/LMG


anoxic waterDCA1T

saturated layer (1 m

in depth) that had

been exclusively

polluted with

50 mg/kg 1,2-DCA

for 30 years




granular sludge and

sp. RPf35Ei



Soil sample from


crop field

sp. KBC1




subsurface soils

sp. B31e3

contaminated with



Ditch sludge

D. hafniense JH1

(mixed with sewage) contaminated

with PCE and halogenated aliphatic


Soil of a former

D. aromaticivorans DSM 19510,

JCM 15765

coal gasification




An anaerobic


mixed culture

sp. PR

enriched from a

bioreactor maintained to perform

dechlorination of

chloroethenes and



Geographic origin Reference

Georgia, USA


et al. (2003)


De Wildeman

et al. (2003)


Pyhäsalmi mine,


Ibaraki, Japan


et al. (2004)



et al. (2006)

Yoshida et al.


Gifu, Japan

Chang et al.


Fletcher et al.


Gliwice, Poland

Kunapuli et al.



Ding et al.


Currently not available. Type strains are indicated by a superscript capital T. NA not available;

NR not reported; PCP pentachlorophenol; PCE tetrachloroethene, DCA dichloroethane

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8 Functional Diversity of Reductive Dehalogenases in the Dehalobacter Genus

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