Diversity, distribution anddivergence of lin genes inhexachlorocyclohexane-degradingsphingomonadsRup Lal, Charu Dogra, Shweta Malhotra, Poonam Sharma and Rinku Pal

Department of Zoology, University of Delhi, Delhi-110 007, India

Two forms of hexachlorocyclohexane (HCH), g-HCH

(lindane) and technical HCH (incorporating a-, b-, g-

and d- isomers), have been used against agricultural

pests and in health programs since the 1940s. Although

all the isomers are present in the milieu, d- and b-HCH

isomers are the most problematic and present a

serious environmental problem. Bacteria that degrade

HCH isomers have been isolated from HCH contami-

nated soils from different geographical locations

around the world (from the family Sphingomonada-

ceae). Interestingly, all these bacteria contain nearly

identical lin genes (responsible for HCH degradation),

which are diverging to perform several catabolic

functions. The organization and diversity of lin genes

have been studied among several sphingomonads, and

they have been found to be associated with plasmids

and IS6100, both of which appear to have a significant

role in their horizontal transfer. The knowledge of the

molecular genetics, diversity and distribution of lin

genes, and the potential of sphingomonads to degrade

HCH isomers, can now be used for developing

bioremediation techniques for the decontamination of

HCH contaminated sites.

Introduction

Hexachlorocyclohexane (HCH) is a cyclic, saturatedhydrocarbon that exists primarily in four isomeric forms:a-, b-, g- and d-HCH. The g-isomer of HCH (commonlyknown as lindane) and technical HCH (which includes a-,b-, g- and d- isomers) have been used extensively againstagricultural pests, and in malaria health programs,worldwide. The indiscriminate use and large-scaleproduction of HCH during the past 50 years has createda serious environmental problem; furthermore, the secretof the unprecedented contamination of our environmentby HCH lies in the unusual chemistry of its production.

The dirty secret of lindane – a chemical of the past

persisting in the future

Commercially, HCH is prepared by the chlorination ofbenzene in the presence of UV, which results in a

Corresponding author: Lal, R. (duzdel@vsnl.com).Available online 13 February 2006

www.sciencedirect.com 0167-7799/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved

mixture of the four isomers: a- (60–70%), b- (5–12%),g- (10–12%) and d- (6–10%) [1]. These isomers differfrom each other by the spatial arrangements of thechlorine atoms on the cyclohexane ring and it is thisarrangement that determines their stability (Figure 1);b-HCH is highly stable because of the presence of thechlorine atoms in equatorial conformation (Figure 1).Only g-HCH has insecticidal potential, and is generallypurified from the rest of the HCH isomers throughseveral concentration and purification steps; theremaining three isomers, also called ‘HCH muck’, arediscarded. Thus, lindane production during the yearshas generated dangerous stockpiles of highly persistenttoxic wastes, which are illegally disposed of in thecountryside, water bodies and agricultural fields inseveral countries [2–5]. Although the use of lindane andtechnical HCH has been banned, or severely restricted,in approximately 50 countries, the presence of HCHresidues is continually reported in different environ-mental compartments [6–11]; furthermore, the spon-taneous dispersal of HCH residues is too slow to be aviable decontamination option.

The anaerobic degradation of HCH isomers was firstreported in 1960s, followed by the isolation of bacteriacapable of metabolizing g-HCH under aerobic conditions– the historical information on the aerobic and anaerobicdegradation of HCH has already been reviewed [12].Although diverse HCH-degrading microorganisms areknown [12,13], members of the family Sphingomonada-ceae appear to have an important role in aerobic HCHdegradation. For example, three HCH-degrading speciesof Sphingobium – Sphingobium japonicum UT26 [14],Sphingobium francense SpC [15] and Sphingobiumindicum B90A [16] (initially classified as three strainsof Sphingomonas paucimobilis) – were isolated fromHCH contaminated soils from Japan, France and India,respectively. Recently, 12 novel HCH-degrading bacterialstrains have been isolated from HCH contaminated sitesat Chemnitz in Germany (DS2, DS2–2 and DS3–1) [17]and Bilbao in northern Spain (g12–7, g16–1, g1–7, a1–2,a4–5, a16–10, a16–12, g16–9 and a4–2) [18]: all theseisolates were also found to be represented bythe members of the family Sphingomonadaceae [17,18].

Review TRENDS in Biotechnology Vol.24 No.3 March 2006

. doi:10.1016/j.tibtech.2006.01.005

Cl

Cl

Cl

ClCl

ClCl

ClCl

Cl

Cl

Cl

Cl

Cl

Cl

ClCl

Cl

ClCl

Cl

Cl

Cl

Cl

H

H

H

HH

H

HH

H

H

HH

HH

H

H

H

H

H

H

H

Cl

Cl

Cl Cl

Cl

ClH

HH

H

H

H

H

H H

α-HCH isomers

γ-HCHβ-HCH

δ-HCH

TRENDS in Biotechnology

Figure 1. Structures of HCH isomers, including the two a-enantiomers. The axial

and equatorial positions of the chlorine atoms are as follows: a, aaaaee; b, eeeeee;

g, aaaeee; d, aeeeee. Abbreviations: a; axial, e; equatorial.

Review TRENDS in Biotechnology Vol.24 No.3 March 2006122

These bacteria degraded a- and g-HCH but therewere differences in their abilities to degrade b-andd-HCH [14,16–18].

Although the molecular genetics of lin genes (associ-ated with HCH degradation) has been explored in-depth,the past five years have witnessed rapid progress in thehorizontal transfer of lin genes, their diversity anddistribution among sphingomonads, and in assigningnew functions to Lin proteins. The focus of this review ison the molecular genetics and evolutionary aspects ofHCH degradation among sphingomonads with an aim todevelop bioremediation technologies.

Complicated pathway of degradation of HCH isomers

Despite the number of organisms known to degradeg-HCH, the degradation pathway has been elucidated indetail only in S. japonicum UT26 [19,20]. The mostimportant steps in the degradation of g-HCH are thedechlorination reactions, as depicted in Figure 2: thedegradation of g-HCH to 2,5-DCHQ (2,5-dichlorohydro-quinone) is referred to as the upstream pathway, and thedegradation of 2,5-DCHQ is referred as the downstreampathway (Figure 2).

(Figure 2. continued) hexachlorocyclohexane; PCCH: pentachlorocyclohexene; 1,4-TCD

DNOL: 2,4,5-trichloro-2,5-cyclohexadiene-1-ol; 2,5-DCP: 2,5-dichlorophenol; 2,5-DDOL: 2

chlorohydroquinone; HQ: hydroquinone; g-HMSA: gamma-hydroxymuconic semialdeh

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Although there are not many reports revealing thepathways of a-, b- and d-HCH degradation, accumulatingevidence suggests that the degradation of b- and d-HCHproceeds by a different pathway than that of a- and g-HCH[21–25] (Lal et al., unpublished data).

Diversity, distribution and divergence of HCH-degrading

(lin) genes among sphingomonads

The catabolic genes associated with the degradation of HCHisomers were initially discovered in S. japonicum UT26, andtermed lin genes [19]. Subsequently, lin genes werediscovered in another HCH-degrading bacterium, Rhodano-bacter lindaniclasticus,whichwas isolated inFrance [26].Sixstructural lin genes (linA–linF) [20,27–31] and one regulat-ory gene (linR) [32] are involved in the complete mineraliz-ation of g-HCH in S. japonicum UT26 (Figure 2; Table 1). Inaddition, a linX gene, encoding a protein that has activitysimilar to that of LinC, was also characterized [29]. Thecomprehensive work on lin genes in S. japonicum UT26, byNagata and co-workers [19], and in S. indicum B90A, by Laland co-workers [15,22], has formed the basis for exploringsimilar genes from S. francense SpC and the recentlydescribed HCH-degrading Sphingomonas strains isolatedin Germany and Spain [15,17].

linA-encoded HCH dehydrochlorinase: a unique but

rapidly diverging gene among HCH-degrading strains

The linA-encoded HCH dehydrochlorinase (LinA)mediates the first two steps of dehydrochlorination ofg-HCH (Figure 2; Table 1). Interestingly, S. indicum B90Awas found to contain two non-identical linA genes(designated as linA1 and linA2) [22]. The nucleotidesequences of linA of S. japonicum UT26 [27], linA2 ofS. indicum B90A [15,22], linA of R. lindaniclasticus [26]and linA of S. francense SpC show 100% sequenceidentity. The linA1 gene of S. indicum B90A, however,differs conspicuously in the C-terminal region (with aminoacids 88% identical to LinA of S. japonicum UT26 andLinA2 of S. indicum B90A [22]), which contained 22nucleotides from an insertion sequence, IS6100, initiallyisolated from the Gram-positive bacterium Mycobacter-ium fortuitum FCI [33]. Of the twelve Sphingomonasstrains isolated in Germany and Spain [17,18], only eightisolates were examined for the presence of lin genes andIS6100 (Table 2) [17]. Sequence analysis revealed that thestrains (DS2, DS2–2, g12–7, g16–1, DS3–1 and a1–2)possess linA genes virtually identical to linA ofS. japonicum UT26 [17]; LinA from g1–7 differed fromthese by only two amino acid positions [17]. Conversely,linA of a4–2 and linA1 of S. indicum B90A are virtuallyidentical to each other but differed from linA ofS. japonicum UT26 at several positions [17].

LinA of S. japonicum UT26 [27], LinA1 and LinA2 ofS. indicum B90A [22] and LinA of DS3–1 [17] were able todegrade a-, g- and d-HCH but not b-HCH [17,22,34], whichwas expected because in all the LinA sequences, the proposedcatalytic residues (His-73 and Asp-25) [35]are conserved [17].

N: 1,3,4,6-tetrachloro-1,4-cyclohexadiene; 1,2,4-TCB: 1,2,4 trichlorobenzene; 2,4,5-

,5-dichloro-2,5-cyclohexadiene-1,4-diol; 2,5-DCHQ: 2,5-dichlorohydroquinone; CHQ:

yde; MA: maleylacetate; GSH, reduced glutathione.

PCCH

1,4-TCDN

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

CHQ HQ

OH

Cl

Cl

HO

OH

Cl

Cl

HO

OH

Cl HO

OH

HO

Cl

Cl

Cl

OH

ClHO CHO

HO

HCl

Cl

Cl Cl

Cl

Cl OH

HCl

HCl

HO MA

O

γ-

Cl

Cl

Cl Cl

Cl

Cl OH

Cl

Cl

Cl

Cl

Cl

OH

Cl

Cl

OH

Cl

Cl

???

OC

β- & δ-HCH

H H

H H

H

H

Cl

Cl

Cl

Cl

Cl

Cl

H H

H

H

H

H

C

Cl

Cl Cl

Cl

Cl

H

H

H

H

H

Cl

Cl

Cl

Cl

Cl

H

H

H

H H

ClCl

Cl

Cl

Cl

?

(-)-α-HCH

(+)-α-HCH

γ-HCH

linA/ linA1/linA2 HCl

linA/ linA1/linA2 HClHCl

Spontaneous 1,2,4-TCB

linB H2O HCl

Spontaneous 2,4,5-DNOL

2,5-DCP

H2O

H2O

linB2,5-DDOL

linCNAD+

NADH + H+

2,5-DCHQ

2GSH

linD

GS-SG+ HCl 2GSH

linD

GS-SG+ HCl

linE /linEb O2

Acylchloride

COOH

Spontaneous

COOH

COOH

γ-HMSA

linE O2

COOH

NADH + H +

NAD+linF

COOH

COOH

β-Ketoadipate

CO2 + H2O

2,3,4,5,6-Pentachlorocyclohexanol Tetrachlorocyclohexanediol

HClH2O

Downstream pathway

β-PCCH

1,2,4-TCB

β-PCCH

Upstream pathway

linB

TRENDS in Biotechnology

Figure 2. Proposed degradation pathways of HCH isomers in Sphingobium japonicum UT26 and Sphingobium indicum B90A. The three different types of dechlorination

reactions sequentially involved in the g-HCH degradation are dehydrochlorination (g-HCH to 1,4-TCDN), hydrolytic dechlorination (1,4-TCDN to 2,5-DDOL) and reductive

dechlorination (2,5-DCHQ to HQ) leading to the central intermediate, chlorohydroquinone (CHQ). This is directly ring-cleaved to produce intermediates that can be further

degraded to b-Ketoadipate, which is metabolized by the b-Ketoadipate pathway. Two dead-end products, 1,2,4-TCB and 2,5-DCP, are produced. g-HMSA is also predicted to

be a dead-end product [19]. linEb, which exhibited significant similarity to LinE (53%) and converted CHQ toMAwas identified in UT26 but does not have an important role in

g-HCH utilization [20]. b- and d-HCH are degraded by LinB to two metabolites, tentatively identified as isomers of pentachlorocyclohexanol and tetrachlorocyclohexanediol,

by hydroxylation reactions in S. indicum B90A [24] (Lal et al., unpublished data). a-HCH-enantiomers are degraded to 1,2,4-TCB via b-PCCH in S. indicum B90A [56]. LinA1

preferentially turns over the (C)-enantiomer of a-HCH, whereas LinA2 prefers the (K)-enantiomer. From each a-HCH enantiomer there are theoretically two possibilities to

eliminate HCl, both leading to the same PCCH enantiomer. The absolute configuration of the b-PCCHs, formed by HCl elimination from a-HCH is shown. Abbreviations: HCH:

Review TRENDS in Biotechnology Vol.24 No.3 March 2006 123

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Table 1. HCH-degrading (lin) genes in Sphingobium indicum B90A, Sphingobium japonicum UT26 and Sphingobium

francense SpC

Nucleotides (amino acids) GCC content (%) Copy no. (stabilityb)

Gene B90A UT26 SpC B90A UT26 SpC Function B90A UT26 SpC Refs

linA1 462 (154) ND ND 52.7 ND ND Dehydro-

chlorinase

2 (CC) 1 (C) 1 (K) [15,19,22]

linA2/linAa 468 (156) 468 (156) 468 (156) 53.9 53.9 53.9

linB 888 (296) 888 (296) 888 (296) 62.5 62.5 62.5 Halidohy-

drolase

1 (CC) 1 (C) 1 (K) [15,19]

linC 750 (250) 750 (250) 750 (250) 64.5 64.3 64.5 Dehydro-

genase

1 (CC) 1 (C) 2 (K) [15,19]

linD 1038

(346)

1038

(346)

1038

(346)

61.8 61.0 61.8 Reductive

dechlori-

nase

1 (C) 1 (C) 1 (CC) [15,19]

linE 963 (321) 963 (321) 963 (321) 60.1 60.1 60.1 Ring-

cleavage

oxygenase

1 (C) 1 (C) 1 (CC) [15,19]

linR 909 (303) 909 (303) 909 (303) 60.3 61.3 60.3 Transcrip-

tional

regulator

1 (C) 1 (C) 1 (C) [15,19]

linX1 750 (250) 750 (250) 750 (250) 64.5 64.5 64.5 Dehydro-

genaselinX2 750 (250) ND ND 64.5 ND ND 3 (CC) 1 (C) 1 (C) [15,19]

linX3 750 (250) ND ND 64.5 ND ND

linF nd 1056

(352)

nd nd 68.1 nd Reductase nd 1 nd [20]

tnpA 792 (264) 792 (264) 792 (264) 61.0 61.0 61.0 Transpo-

sase

11 (C) 5 nd 6 (K) [15]

alinA represents the linA gene of UT26 and linA of SpC; bCC: highly stable; C: stable; K: unstable. Abbreviations: ND: not detected; nd: not determined.

Review TRENDS in Biotechnology Vol.24 No.3 March 2006124

Subsequent analysis revealed that LinA dehydrochlorinatesstereoselectively at a 1,2-biaxial pair of hydrogen andchlorine [36], which are present in a-, g- and d-HCH(Figure 1). Because b-HCH and d-PCCH [36] lack a 1,2-biaxial HCl group they are not subject to LinA activity.Additionally,LinA(16.5 kDa)doesnot requirecofactors for itsactivity and is, therefore, a unique dehydrochlorinase, and isdistinct from the two other previously reported dehydro-chlorinases: DDT dehydrochlorinase from Musca domestica(which requires glutathione) and 3-chloro-D-alanine dehy-drochlorinase from Pseudomonas putida (which requirespyridoxal 50-phosphate).

Table 2. Presence and genetic organization of lin genes and IS6100

lin genes

Strain/

location

linA1 linA2/

linAa

linB linC linDER linX1 linX2

B90A/

India

C C C C C C C

UT26/

Japan

K C C C C C K

SpC/

France

K C C C C C K

DS2/

Germany

K C C C C C C

DS2-2/

Germany

K C C C C C C

DS3-1/

Germany

K C C C C C C

a1-2/Spain K C C C C C C

a4-2/Spain C K C C C C C

g1-7/Spain K C C C C C C

g12-7/

Spain

K C C C K C C

g16-1/

Spain

K C C C C C C

alinA represents the linA gene of UT26 and linA of SpC. C: present; K: absent; nd: not

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linB-encoded halidohydrolase performs multiple

functions and belongs to a/b-hydrolase family

The linB-encoded halidohydrolase (LinB) is responsiblefor the hydrolytic dechlorination of 1,4-TCDN (1,3,4,6-tetrachloro-1,4-cyclohexadiene) to 2,5-DDOL (2,5-dichloro-2,5-cyclohexadiene-1,4-diol) [28] (Figure 2;Table 1). Comparison of the amino acid sequences ofLinB in three species showed 97%, 97% and 98%similarity between S. indicum B90A: S. francenseSpC; S. indicum B90A: S. japonicum UT26; andS. francense SpC: S. japonicum UT26, respectively(Lal et al., unpublished data). Preliminary studies also

in HCH-degrading sphingomonads

Organization of lin genes and their

association with IS6100

linX3 (linX,linX2,

linA/IS6100)

(IS6100/

linB/IS6100)

(IS6100/

linC/IS6100)

Refs

C C/C C/C/C C/C/C [15,17]

K C/K K/C/C K/C/C [17,19]

K C/C nd/C/C nd/C/nd [15]

nd C/K K/C/C K/C/K [17]

nd C/K K/C/C K/C/K [17]

nd C/C C/C/C C/C/C [17]

nd C/C K/C/C K/C/C [17]

nd C/C C/C/C K/C/C [17]

nd C/K K/C/C K/C/C [17]

nd C/K C/C/C K/C/K [17]

nd C/K C/C/C K/C/K [17]

determined.

Review TRENDS in Biotechnology Vol.24 No.3 March 2006 125

indicated strong homology among the linB genes ofR. lindaniclasticus and S. japonicum UT26 [26]. Thepresence of linB genes has also been demonstrated inrecently isolated HCH-degrading sphingomonads(Table 2) [17].

Unlike LinA, which seems to be a unique dehydro-chlorinase, LinB is a haloalkane dehalogenase of the a/b-hydrolase family of enzymes that shows significantsimilarity to three types of a/b hydrolase fold enzymes:haloalkane dehalogenase (DhlA) from Xanthobacter auto-trophicus GJ10; haloacetate dehalogenase (DehH1) fromMoraxella sp. B; and serine hydrolases represented by2-hydroxymuconic semialdehyde hydrolase (DmpD) fromPseudomonas sp. CF600. The 32 kDa LinB protein hasrelatively broad substrate specificity [37,38] and, thus, hasbeen subjected to crystallographic [39,40], mutagenesis[41] and computational [42] studies.

In addition to mediating the second step in thedegradation of g-HCH in S. japonicum UT26(Figure 2), LinB has been reported, recently, totransform b-HCH to 2,3,4,5,6-pentachlorocyclohexanol(PCHL) [43]. PCHL has lower hydrophobicity andlower chemical stability than b-HCH, and the bacteriathat degrade and use it might exist in the pollutedenvironment, enabling the complete degradation ofb-HCH by a combination of biological pathways.Interestingly, LinB has been found to be involved inthe degradation of b- and d-HCH in S. indicum B90A,S. francense SpC and S. japonicum UT26 (Lal et al.,unpublished data), although the ability to degradeb-HCH and d-HCH differed. Based on a previousreport [24], and coupled with the studies carried outin our laboratory (Lal et al., unpublished data), theinitial pathway for the degradation of b- and d-HCHin S. indicum B90A is depicted in Figure 2.

linC and linX genes in HCH-degrading sphingomonads

The linC gene encodes 2,5-DDOL dehydrogenase (LinC),which is responsible for the conversion of 2,5-DDOL (2,5-dichloro-2,5-cyclohexadiene-1,4-diol) to 2,5-DCHQ (2,5-dichlorohydroquinone) [29] (Figure 2; Table 1). Inaddition, a linX gene, encoding a protein that has activitysimilar to that of LinC, was also characterized [29]. Thededuced amino acid sequence of LinX revealed 33.1%identity with that of LinC [19]; furthermore, LinC andLinX (both 28 kDa) showed significant similarity tomembers of the short-chain alcohol dehydrogenase super-family. Unlike single copies of linX and linC genes found inS. japonicum UT26 [29], three copies of linX [15] and twocopies of linC have been found in S. indicum B90A andS. francense SpC, respectively. One copy of linC fromS. francense SpC was sequenced and showed 99% DNAsequence similarity to linC of S. indicum B90A [15] andS. japonicum UT26 [29] (GenBank accession no.DQ111068). The two copies of linX (designated linX1 andlinX3) in S. indicum B90A showed 99% DNA sequencesimilarity with linX of S. japonicum UT26, whereas linX2differed, conspicuously, from the other copies of linX: theintact linX2 ORF exhibited only 66% similarity to linX1,linX3 or linX [15]. The presence of linC, linX1 and linX2

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has also been shown in recently isolated HCH-degradingsphingomonads (Table 2) [17].

Downstream pathway genes among HCH-degrading

strains

Four genes, linD [30], linE [31], linR [32] and linF [20],which encode reductive dechlorinase (LinD, 38.4 kDa),ring-cleavage oxygenase (LinE, 36.0 kDa), a transcrip-tional regulator (LinR, 33.6 kDa) and maleyl acetatereductase (LinF, 38.0 kDa), respectively, are associatedwith the downstream pathway of g-HCH degradation(Figure 2; Table 1). The nucleotide sequences of the threegenes (linDER) in S. japonicum UT26, S. indicum B90A[15] and S. francense SpC were 99–100% identical. Thepresence of linDER was also detected in all the isolatesfrom Germany and Spain, except in g12–7 [17] (Table 2),suggesting that these strains employ similardownstream pathways.

LinD, LinE and LinF were significantly similar to theenzymes that are involved in pentachlorophenol degra-dation in Sphingomonas chlorophenolica ATCC 39723(PcpC, PcpA and PcpE, respectively) [20,30,31]. LinE alsoshowed a low level of similarity to meta-cleavagedioxygenases, and was identified as a novel type of theseenzymes [31]. LinR showed similarity to LysR-typetranscriptional regulators (LTTRs) [32,44]. Theexpression of linD and linE was induced by LinR in thepresence of 2,5-dichlorohydroquinone (2,5-DCHQ), chloro-hydroquinone (CHQ) and hydroquinone (HQ) inS. japonicum UT26 [32]. These results indicated thatLinR is a positive transcriptional regulator for theexpression of linD and linE.

Horizontal journey of lin genes and their on-going

evolution

Evidence is available to implicate horizontal gene-transfer(HGT), mediated by mobile genetic elements (MGEs), inthe recruitment and establishment of HCH catabolicpathways, or genes, in sphingomonads.

HCH-degrading strains from different geographical

locations are members of the family

Sphingomonadaceae

The HCH-degrading bacterial strains isolated from India,Japan, France, Germany and Spain have not only beenanalyzed for the presence of lin genes but have also beensubjected to phylogenetic analysis. The taxonomicalpositions of S. indicum B90A, S. japonicum UT26 andS. francense SpC, initially thought to be three strains ofSphingomonas paucimobilis, have been ascertained,recently, and are now classified as three distinct species(i.e. Sphingobium indicum sp. nov, Sphingobium japoni-cum sp. nov and Sphingobium francense sp. nov, respect-ively) [45]. Based on 16S rRNA gene analysis, the recentlyisolated strains from HCH dumpsites in Germany andSpain were found to be members of the family Sphingo-monadaceae [17,18]. Although Boltner et al. [17] andMohn et al. [18] classified them as Sphingomonas strains,according to the new scheme of classification [46] theseisolates fall in clades represented by Sphingopyxis, Novo-sphingobium,Sphingobium andSphingomonas (Figure 3);

Sphingobium yanoikuyae GIFU 9882T (D84526)

Sphingomonas ursincola DSM 9006T (AB024289)

1000

262

367

326

882

333 917

269

747

966

845

508

433

390

336

444

1000

935

981

278

140

756

501

439

742

364

459

701

978

820

991

988

896

982

1000

590

911

1000

1000

486

432

616

991

945

1000

781

1000

0.1

Sphingomonas pruni IFO 15498T (Y09637)Sphingomonas mali IFO 10550T (Y09638)Sphingomonas asaccharolytica IFO 10564T (Y09639)Sphingomonas oligophenolica DSM 17107T (AB018439)

Sphingomonas echinoides DSM 1805T (AJ012461)Sphingomonas koreensis DSM 15582T (AF131296) Sphingomonas melonis DSM 14444T (AB055863)Sphingomonas aquatilis DSM 15581T (AF131295)

Sphingomonas aerolata DSM 14746T (AJ429240) Sphingomonas faeni DSM 14747T (AJ429239)

Sphingomonas aurantiaca DSM 14748T (AJ429236) Sphingomonas phyllosphaerae LMG 21958T (AY453855)

Sphingomonas adhaesiva DSM 7418T (D16146) Sphingomonas trueperi LMG 2142T (X97776)Sphingomonas pituitosa DSM 13101T (AJ243751)

Novosphingobium capsulatum GIFU 11526T (D84532)Sphingomonas paucimobilis ATCC 29837T (U37337)

Sphingomonas sanguinis DSM 13885T (D13726) Sphingomonas parapaucimobilis JCM 7510T (D84525)

Sphingomonas roseiflava IAM 14823T (D84520) Sphingomonas yabuuchiae DSM 14562T (AB071955)

Sphingopyxis terrae IFO 15098T (D84531)Sphingomonas wittichii DSM 6014T (AB021492)

Sphingomonas sp. α1-2 (AY771794)Sphingomonas sp. α4-5 (AY771798)Sphingomonas sp. α4-2 (AY771797)

Sphingomonas suberifaciens IFO 15211T (D13737)Sphingomonas xenophaga DSM 6383T (X94098)

Sphingomonas cloacae DSM 14926T (AB040739)

Sphingobium amiense JCM 11777T (AB047364) Sphingobium herbicidovorans DSM 11019T (AB022428) Sphingobium japonicum CCM 7287T (AF039168) Sphingobium indicum CCM 7286T (AY519129)Sphingobium francense CCM 7288T (AY519130)Sphingobium chungbukense DSM 16638T (AF159257)

Sphingobium chlorophenolicum ATCC 33790T (X87161)Sphingomonas sp. α16-10 (AY771795)Sphingomonas sp. α16-12 (AY771796)Sphingomonas sp. DS2-2 (AJ871277)

Sphingomonas sp. DS2 (AJ871276)

902

706

727

542

Novosphingobium subterraneum DSM 12447T (U20773)Novosphingobium aromaticivorans DSM 12444T (U20756)Novosphingobium taihuense JCM 12465T (AY500142)

Novosphingobium hassiacum DSM 14552T (AJ416411) Novosphingobium lentum DSM 13663T (AJ303009) Novosphingobium stygium DSM 12445T (U20775) Novosphingobium tardaugens DSM 16702T (AB070237)

Novosphingobium subarcticum DSM 10700T (X94102)

Sphingopyxis baekryungensis DSM 16222T (AY608604) Sphingopyxis flavimaris DSM 16223T (AY554010)

Sphingopyxis witflariensis DSM 14551T (AJ416410)Sphingopyxis alaskensis DSM 13593T (AY337601)

Sphingopyxis macrogoltabida IFO 15033T (D84530)Sphingopyxis chilensis DSM 14889T (AF367204) Sphingomonas taejonensis DSM 15583T (AF131297)Sphingomonas sp. DS3-1 (AJ871278)

Rhodanobacter lindaniclasticus RP5557T (AF039167)

946

514

695

534

304

1000

364

997

1000

578

Sphingomonas sp. γ16-9 (AY771801)Sphingomonas sp. γ16-1 (AY771800)Sphingomonas sp. γ12-7 (AY771799)

Sphingomonas sp. γ1-7 (AY771802)

TRENDS in Biotechnology

Figure 3. Phylogenetic tree based on nearly complete 16S rRNA gene sequences (1337 aligned positions) showing relative taxonomic positions of HCH-degrading

sphingomonads isolated from Japan, India, France, Germany and Spain (in bold). The tree was constructed by neighbour-joining method and rooted by using

Review TRENDS in Biotechnology Vol.24 No.3 March 2006126

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linX1

linA2

linA

B90A

B90A

Sp+

Sp+

B90A

linC

linC

linC

‘orfM

Sp+

linDB90A

Sp+

linX1 linX2 linA1

linA2

+

linD

orfH linX2 linA1 IS6100 orfJ’

linX ORFUP linA

UT26

orfH linX3 ORFUP IS6100

IS6100

4.02.00.0 6.0

4.02.00.0 6.0

UT26linB IS6100

IS6100 linB IS6100

linB IS6100 2.01.51.00.50.0

UT26

B90A

IS6100 IS6100 ‘orfN

‘orfM

4.02.00.0 6.0

‘orf8 linR linE orf2 orf4 linD ‘orf5

orf3

‘orf1 linR linE orf2

orf3

orf4

linD‘orf1 linR linE orf2

orf3

orf4

‘orf5

‘orf5

IS6100

IS6100

UT26

IS6100 IS6100 orfJ orfK orfL

linX3 IS6100

linB IS6100

IS6100 linC IS6100 orfL

IS6100 orf2 orf3 orf4linE linR

(a)

(b)

(c)

(d)

(e)

IS6100

Figure 4. (a) ‘Mosaic’ organization of linA gene in Sphingobium indicum B90A (linA1 and linA2), Sphingobium japonicum UT26 (linA) and S. francense SpC (linA). (b)

Comparison of the organization of linB gene in S. japonicum UT26, S. indicum B90A and S. francense SpC. (c) Organization of linC gene in S. japonicum UT26, S. indicum

B90A and S. francense SpC. (d)Organization of linDER gene in S. japonicumUT26, S. indicum B90A and S. francense SpC. (e) Six transcriptional units (linX1X2A1, linX3A2,

linB, linC, linDE and linR) encoding the g-HCH degradation pathway in S. indicum B90A. linA (combination of linA1 and linA2), linB, linC and linX are constitutively expressed.

The only known regulatable promoter among the lin genes is in front of linE and is activated by LinR. The function of linX gene is not known but, based on sequence

homology, it was predicted to be a dehydrogenase, perhaps similar in function to LinC. Arrows denote the direction of transcription. Where IS6100 is depicted by a box,

direction of transcription is not known.

Review TRENDS in Biotechnology Vol.24 No.3 March 2006 127

however, the validity of the new genera, indicated inFigure 3, remains controversial thus far [47]. Never-theless, similar to S. indicum B90A, S. japonicum UT26and S. francense SpC, the strains isolated from Germanyand Spain also appear to be distinct species belonging tothe family Sphingomonadaceae.

lin genes and their association with IS6100 and plasmids:

the process of evolution continues

The association of IS6100 with lin genes among all thesphingomonads [15,17] implies that IS6100 is almostcertainly involved in the HGT, presumably by increasingthe potential of further exchange of the genes betweendifferent hosts and replicons. The IS6100 element (880 bp)belongs to the IS6 family [48], and has an extremely broadhost range [49–52]. Recently, a copy of the IS6100 wasfound to be associated with the second chlorocatecholcatabolic gene cluster in Sphingomonas sp. TFD44 [53]and Sphingomonas herbicidovorans MH [54].

The linA gene was flanked by a copy of IS6100 inS. indicum B90A, S. francense SpC, DS3–1, a1–2 and a4–2 [15,17] (Table 2; Figure 4a). Similarly, the linB gene wasflanked by a copy of IS6100 in S. indicum B90A,S. francense SpC, S. japonicum UT26 and all of theeight isolates from Germany and Spain [15,17] (Table 2;

(Figure 3. continued) Rhodanobacter lindaniclasticus as an out-group. The numbers at no

0.1 nucleotide substitution per nucleotide position. The GenBank accession number for

validly described species belonging to the genera Sphingomonas, Sphingobium, Novos

tree.

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Figure 4b). Moreover, linB (in DS3–1, a4–2, g12–7, g16–1and S. indicum B90A) [17] and linC (in S. indicum B90A),in addition to DS3–1 [15,17] (Figure 4c), were flanked bytwo IS6100 copies, possibly making composite transpo-sons. Furthermore, linC was flanked by a copy of IS6100in S. japonicum UT26 (Figure 4c), g1–7 and a1–2 [17](Table 2). Similarly, linD gene was flanked by a copy ofIS6100 in S. indicum B90A [15] and S. francense SpC(Figure 4d). Spontaneous deletion of linD and linE fromS. indicum B90A, and of linA from S. francense SpC, wasobserved and was associated either with the deletion of acopy of IS6100 or changes in IS6100 profiles [15].

In addition, the overall organization of catabolic genesinvolved in the degradation of HCH isomers revealed thatlin genes are not organized in co-ordinately regulatedoperons. Instead, at least six different transcriptionalunits forming the g-HCH degradation pathway inS. indicum B90A (linX1X2A1, linX3A2, linB, linC, linDEand linR) are proposed (Figure 4e). linA, linB and linCwere constitutively expressed [19,21], whereas linD andlinE could only be induced by g- and a-HCH, and not byb- and d-HCH, in S. indicum B90A [21]. Our recent studieshave also revealed that lin genes are not uniformlydistributed on the chromosome but are also present onplasmids in S. indicum B90A, S. japonicum UT26 and

de represent bootstrap values (based on 1000 resamplings). The scale bar indicates

the 16S rRNA gene sequence of each reference species is shown in parenthesis. 51

phingobium and Sphingopyxis were analyzed when constructing the phylogenetic

Review TRENDS in Biotechnology Vol.24 No.3 March 2006128

S. francense SpC (Lal et al., unpublished data). Thus,IS6100 and plasmids (probably conjugative) appear tohave a significant role in HGT of lin genes amongsphingomonads. The conjugative transfer of degradativeplasmids in Sphingomonas strains was also recentlydemonstrated [55].

Diversity in lin genes leads to divergence in their function

The lin genes identified in strains isolated from Japan,India, France, Germany and Spain appear to be highlyconserved [15,17,19]. However, multiple copies of some lingenes have been detected in S. indicum B90A [15] andS. francense SpC (Table 1). The tendency of catabolic genesto undergo genetic rearrangements (duplications andinsertions) is due to the presence of transposable elementsand, therefore, can be attributed to IS6100 present in theorganisms described. Generally, gene duplication providesan opportunity for one of the duplicated genes to accumulatemutations and eventually diverge to an extent where itperforms a new function. This is evident from theenantioselective transformation of the (C)-enantiomer ofa-HCH by LinA1 and (-)-enantiomer of a-HCH by LinA2 inS. indicum B90A [56] (Figure 2). The enantioselectivetransformation of a-HCH by LinA1 and LinA2 provides thefirst evidence of the evolution of enantioselective genes inS. indicum B90A.

Acquisition of lin genes from unknown host(s)

The detection of similar HCH-degrading genes and genecombinations in several sphingomonads [15,17,19] can beexplained either by independent recruitment of genes orby long distance transport of bacteria combined with theHGT of all the genes. Evidence is accumulating[15,17,19,45] that lin genes are not native to S. indicumB90A, S. japonicum UT26, and S. francense SpC and havecome from unknown host(s), independently, in most of thesphingomonads. For instance, the GCC content of all linAgenes reported so far is lower (w53%) than the GCCcontent of the genes ascribed for the Sphingobium spp.genome (Table 1) [15,17,19,22,26]. Moreover, there are nohomologues of linA in GenBank, except for a few openreading frames with low sequence identity and unknownfunctions. Based on the structural comparisons, it wasproposed that LinA might have evolved from a dehydra-tase enzyme [35]. If, indeed, linA has evolved and spreadrecently – following the widespread release of HCH sincethe 1940s – the number of mutations that have accumu-lated in different strains [15,17,22,27] in such a short timescale might indicate an unusually high mutation rate.Although the GCC content of linB, linC and linDE is closeto that of Sphingobium spp. genome (w65%), theirpresence on plasmids and/or their association withIS6100 [15], in addition to their absence from non-HCH-degrading organisms [45], indicates lateral acquisition ofthese genes as well.

Although it is not clear what makes the members of thefamily Sphingomonadaceae so adaptable to HCH degra-dation, one factor that probably contributes towards this isthe presence of glycosphingolipids instead of lipopolysac-charides in the outer membrane [57]: this provides ahighly hydrophobic surface, which might facilitate the

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assimilation of hydrophobic compounds such as HCH [58].The presence of lin genes and IS6100 on plasmids addsanother factor that might have contributed to theextraordinary spread of lin genes among HCH-degrading sphingomonads.

MGEs as a tool in bioremediation of HCH

The accumulating information on the genetic, molecularand physiological aspects of HCH degradation, coupledwith the thorough knowledge of MGEs functions innature, is important for the development of bioremedia-tion method(s). Because catabolic genes (lin genes) arepresent on MGEs (plasmids and IS6100), the introductionof HCH-degrading bacteria into the indigenous bacterialpopulation by bioaugmentation can be attempted,whereby the genes spread by HGT. It is evident from theliterature reviewed that S. indicum B90A, whose geneticshave now been well characterized [15,21,22,45,56], has abetter HCH-degrading capacity compared with all thesphingomonads discussed so far. Furthermore, S. indicumB90A has evolved a stable pathway for the degradation ofHCH isomers (a-, b-, g- and d-HCH) because the loss oflinA and, to a lesser extent, of other lin genes wasfrequently observed in all the sphingomonads except inS. indicum B90A [15,17,22,59,60]. Thus, sphingomonadssuch as S. indicum B90A can be used to develop aneffective bioremediation technology.

Future prospects

Although our understanding of the diversity anddistribution of lin genes in sphingomonads hasadvanced, several interesting aspects, including thoserelated to finding the original host(s) containing lingenes, exploring b- and d-HCH-degrading pathway(s)and exploiting the potential of these organisms todecontaminate HCH isomers, are challenges for thefuture. New information on the topic will undoubtedlybecome available through the analysis of the sequencesof the plasmids associated with lin genes and thegenomes of these organisms.

Acknowledgements

Part of the work was supported by grants under the Indo–SwissCollaboration in Biotechnology (ISCB) from the Swiss Agency forDevelopment and Cooperation (SDC), Switzerland, and Department ofBiotechnology (DBT), India. C.D., S.M. and P.S. gratefully acknowledgeCSIR-UGC, Government of India, for providing the research fellowships.We thank C. Holliger, J.R. van der Meer, R.K. Jain, Banwari Lal, Hans-Peter Kohler and Hans-Rudolf Buser for their input during thepreparation of this manuscript.

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