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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. ([email protected]).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
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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
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H
H
H
H
Cl
Cl
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H
H
H
H H
ClCl
Cl
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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.
References
1 Willett, K.L. et al. (1998) Differential toxicity and environmental fatesof hexachlorocyclohexane isomers. Environ. Sci. Technol. 32,2197–2207
2 Oliveira, R.M. et al. (2003) Residual concentration of hexachlorocy-clohexane in a contaminated site in Cidade dos Meninos, Duque deCaxias, Rio de Janeiro, Brazil, after calcium oxide treatment. Cad.Saude Publica 19, 447–453
3 Osterreicher-Cunha, P. et al. (2003) HCH distribution and microbialparameters after liming of a heavily contaminated soil in Rio deJaneiro. Environ. Res. 93, 316–327
Review TRENDS in Biotechnology Vol.24 No.3 March 2006 129
4 Prakash, O. et al. (2004) Residues of hexachlorocyclohexane (HCH)isomers in soil and water samples from Delhi and adjoining areas.Curr. Sci. 87, 73–77
5 Willem, J.K. and Wollent, I. (2005) Inventories of obsolete pesticidestocks in Central and Eastern Europe. In 7th International HCH andPesticides Forum (Elbestawy, E. et al., eds), pp. 37–39, SustainableDevelopment and Ecological Research Center, Ukraine and Inter-national HCH and Pesticides Associations
6 Popp, P. et al. (2000) Chlorobenzenes and hexachlorocyclohexanes(HCHs) in the atmosphere of Bitterfeld and Leipzig (Germany).Chemosphere 41, 849–855
7 Bakore, N. et al. (2004) Organochlorine pesticide residues in wheatand drinking water samples from Jaipur, Rajasthan, India. Environ.Monit. Assess. 98, 381–389
8 Bidleman, T.F. and Leone, A.D. (2004) Soil–air exchange of organo-chlorine pesticides in the Southern United States. Environ. Pollut.128, 49–57
9 Blasco, C. et al. (2004) Determination of organochlorine pesticideresidues in honey from the central zone of Portugal and the Valenciancommunity of Spain. J. Chromatogr. A 1049, 155–160
10 Nigam, U. and Siddiqui, K.J. (2001) Organochlorine insecticidesresidues in dairy milk samples collected in Lucknow, India. Bull.Environ. Contam. Toxicol. 66, 678–682
11 Bhatnagar, V.K. et al. (2004) Levels of DDT, HCH and HCB residues inhuman blood in Ahmedabad, India. Bull. Environ. Contam. Toxicol.72, 261–265
12 Phillips, T.M. et al. (2005) Biodegradation of hexachlorocyclohexane(HCH) by microorganisms. Biodegradation 16, 363–392
13 Kumar, M. et al. (2005) Enhanced biodegradation of b-hexachlor-ocyclohexane in the presence of a- and g-isomers in contaminatedsoils. Environ. Sci. Technol. 39, 4005–4011
14 Imai, R. et al. (1989) Dehydrochlorination of g-hexachlorocyclohexane(g-BHC) by g-BHC-assimilating Pseudomonas paucimobilis. Agric.Biol. Chem. 53, 2015–2017
15 Dogra, C. et al. (2004) Organization of lin genes and IS6100 amongdifferent strains of hexachlorocyclohexane-degrading Sphingomonaspaucimobilis – evidence for horizontal gene transfer. J. Bacteriol. 186,2225–2235
16 Sahu, S.K. et al. (1990) Degradation of alpha-, beta-, gamma-hexachlorocyclohexane by a soil bacterium under aerobic conditions.Appl. Environ. Microbiol. 56, 3620–3622
17 Boltner, D. et al. (2005) 16S rDNA phylogeny and distribution of lingenes in novel hexachlorocyclohexane-degrading Sphingomonasstrains. Environ. Microbiol. 7, 1329–1338
18 Mohn, W.W. et al. (2005) Distribution and phylogeny of hexachlor-ocyclohexane-degrading bacteria in soils from Spain. Environ.Microbiol. 8, 60–68
19 Nagata, Y. et al. (1999) Complete analysis of genes and enzymes forg-hexachlorocyclohexane degradation in Sphingomonas paucimobilisUT26. J. Ind. Microbiol. Biotechnol. 23, 380–390
20 Endo, R. et al. (2005) Identification and characterization of genesinvolved in the downstream degradation pathway of g-hexachlorocy-clohexane in Sphingomonas paucmobilis UT26. J. Bacteriol. 187,847–853
21 Suar, M. et al. (2004) Dynamics of multi lin gene expression inSphingomonas paucimobilis B90A in response to different HCHisomers. Appl. Environ. Microbiol. 70, 6650–6656
22 Kumari, R. et al. (2002) Cloning and characterization of lin genesresponsible for degradation of hexachlorocyclohexane isomers inSphingomonas paucimobilis strain B90. Appl. Environ. Microbiol.68, 6021–6028
23 Trantirek, L. et al. (2001) Reaction mechanism and stereochemistry ofg-hexachlorocyclohexane dehydrochlorinase LinA. J. Biol. Chem. 276,7734–7740
24 Sahu, S.K. et al. (1995) Mineralization of a-, g- and b-isomers ofhexachlorocyclohexane by a soil bacterium under aerobic conditions.J. Agric. Food Chem. 43, 833–837
25 Nagata, Y. et al. (2005) Degradation of b-hexachlorocyclohexane byhaloalkane dehalogenase LinB from Sphingomonas paucimobilisUT26. Appl. Environ. Microbiol. 71, 2183–2185
26 Thomas, J.C. et al. (1996) Isolation and characterization of a novelg-hexachlorocyclohexane-degrading bacterium. J. Bacteriol. 178,6049–6055
www.sciencedirect.com
27 Imai, R. et al. (1991) Molecular cloning of a Pseudomonas paucimobilisgene encoding a 17-kilodalton polypeptide that eliminatesHCl molecules from g-hexachlorocyclohexane. J. Bacteriol. 173,6811–6819
28 Nagata, Y. et al. (1993) Cloning and sequencing of a dehalogenase geneencoding an enzyme with hydrolase activity involved in thedegradation of g-hexachlorocyclohexane in Pseudomonas paucimobi-lis. J. Bacteriol. 175, 6403–6410
29 Nagata, Y. et al. (1994) Cloning and sequencing of a 2,5-dichloro-2,5-cyclohexadiene-1,4-diol-dehydrogenase gene involved in the degra-dation of g-hexachlorocyclohexane in Pseudomonas paucimobilis.J. Bacteriol. 176, 3117–3125
30 Miyauchi, K. et al. (1998) Cloning and sequencing of a 2,5-dihydrochloroquinone reductive dehalogenase gene whose product isinvolved in the degradation of g-hexachlorocyclohexane by Sphingo-monas paucimobilis. J. Bacteriol. 180, 1354–1359
31 Miyauchi, K. et al. (1999) Cloning and sequencing of a novel meta-cleavage dioxygenase gene whose product is involved in degradation ofg-hexachlorocyclohexane in Sphingomonas paucimobilis. J. Bacteriol.181, 6712–6719
32 Miyauchi, K. et al. (2002) Cloning and characterization of linR,involved in regulation of downstream pathway for g-hexachlorocyclo-hexane degradation in Sphingomonas paucimobilis UT26. Appl.Environ. Microbiol. 68, 1803–1807
33 Martin, C. et al. (1990) Transposition of an antibiotic resistanceelement in mycobacteria. Nature 345, 739–743
34 Nagata, Y. et al. (1993) Purification and characterization ofg-hexachlorocyclohexane (g-HCH) dehydrochlorinase (LinA) fromPseudomonas paucimobilis. Biosci. Biotechnol. Biochem. 57,1582–1583
35 Nagata, Y. et al. (2001) Identification of protein fold and catalyticresidues of g-hexachlorocyclohexane dehydrochlorinase LinA. Pro-teins 45, 471–477
36 Trantirek, L. et al. (2001) Reaction mechanism and stereochemistry ofg-hexachlorocyclohexane dehydrochlorinase LinA. J. Biol. Chem. 276,7734–7740
37 Janssen, D.B. (2004) Evolving haloalkane dehalogenases. Curr. Opin.Chem. Biol. 8, 150–159
38 Nagata, Y. et al. (1997) Purification and characterization of haloalkanedehalogenase of a new substrate class from a g-hexachlorocyclo-hexane-degrading bacterium, Sphingomonas paucimobilis UT26.Appl. Environ. Microbiol. 63, 3707–3710
39 Oakley, A.J. et al. (2004) Crystal structure of haloalkane dehalogenaseLinB from Sphingomonas paucimobilis UT26 at 0.95 A resolution:dynamics of catalytic residues. Biochemistry 43, 870–878
40 Streltsov, V.A. et al. (2003) Haloalkane dehalogenase LinB fromSphingomonas paucimobilis UT26: X-ray crystallographic studies ofdehalogenation of brominated substrates. Biochemistry 42,10104–10112
41 Chaloupkova, R. et al. (2003) Modification of activity and specificityof haloalkane dehalogenase from Sphingomonas paucimobilis UT26by engineering of its entrance tunnel. J. Biol. Chem. 278,52622–52628
42 Otyepka, M. and Damborsky, J. (2002) Functionally relevant motionsof haloalkane dehalogenases occur in the specificity-modulating capdomains. Protein Sci. 11, 1206–1217
43 Nagata, Y. et al. (2005) Degradation of b-hexachlorocyclohexane byhaloalkane dehalogenase LinB from Sphingomonas paucimobilisUT26. Appl. Environ. Microbiol. 71, 2183–2185
44 Schell, M.A. (1993) Molecular biology of the LysR family oftranscriptional regulators. Annu. Rev. Microbiol. 47, 597–626
45 Pal, R. et al. (2005) Hexachlorocyclohexane-degrading bacterialstrains Sphingomonas paucimobilis B90A, UT26 and SpC, havingsimilar lin genes, represent three distinct species, Sphingobiumindicum sp. nov., Sphingobium japonicum sp. nov. and Sphingobiumfrancense sp. nov., and reclassification of [Sphingomonas] chungbu-kensis as Sphingobium chungbukense comb. nov. Int. J. Syst. Evol.Microbiol. 55, 1965–1972
46 Takeuchi, M. et al. (2001) Proposal of the genus Sphingomonassensu stricto and three new genera, Sphingobium, Novosphingo-bium and Sphingopyxis, on the basis of phylogenetic andchemotaxonomic analyses. Int. J. Syst. Evol. Microbiol. 51,1405–1417
Review TRENDS in Biotechnology Vol.24 No.3 March 2006130
47 Yabuuchi, E. et al. (2002) Emendation of the genus SphingomonasYabuuchi et al. 1990 and junior objective synonymy of the species ofthree genera, Sphingobium, Novosphingobium and Sphingopyxis, inconjunction with Blastomonas ursincola. Int. J. Syst. Evol. Microbiol.52, 1485–1496
48 Mahillon, J. and Chandler, M. (1998) Insertion sequences. Microbiol.Mol. Biol. Rev. 62, 725–774
49 Tauch, A. et al. (2002) The 27.8-kb R-plasmid pTET3 fromCorynebacterium glutamicum encodes the aminoglycoside adenyl-transferase gene cassette aadA9 and the regulated tetracycline effluxsystem Tet 33 flanked by active copies of the widespread insertionsequence IS6100. Plasmid 48, 117–129
50 Hall, R. et al. (1994) Integrons found in different locations haveidentical 5 0 ends but variable 3 0 ends. J. Bacteriol. 176, 6286–6294
51 Sundin, G.W. and Bender, C.L. (1995) Expression of the strA–strBstreptomycin resistance genes in Pseudomonas syringae and Xantho-monas campestris and characterization of IS6100 in X. campestris.Appl. Environ. Microbiol. 61, 2891–2897
52 Kato, K. et al. (1994) Insertion sequence IS6100 on plasmidpOAD2, which degrades nylon oligomers. J. Bacteriol. 176,1197–1200
53 Thiel, M. et al. (2005) Two unusual chlorocatechol catabolic geneclusters in Sphingomonas sp. TFD44. Arch. Microbiol. 183,80–94
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54 Muller, T.A. et al. (2004) Genetic analysis of phenoxyalkanoic aciddegradation in Sphingomonas herbicidovorans MH. Appl. Environ.Microbiol. 70, 6066–6075
55 Basta, T. et al. (2004) Detection and characterization of conjugativedegradative plasmids in xenobiotic-degrading Sphingomonas strains.J. Bacteriol. 186, 3862–3872
56 Suar, M. et al. (2005) Enantioselective transformation of a-hexachlor-ocyclohexane (a-HCH) by the dehydrochlorinases, LinA1 and LinA2,from the soil bacterium Sphingomonas paucimobilis B90A. Appl.Environ. Microbiol. 71, 8514–8518
57 White, D.C. et al. (1996) The genus Sphingomonas: physiology andecology. Curr. Opin. Biotechnol. 7, 301–306
58 Kawahara, K. et al. (1999) Chemical structure and function ofglycosphingolipids of Sphingomonas spp. and their distributionamong members of the a-4 subclass of Proteobacteria. J. Ind.Microbiol. Biotechnol. 23, 408–413
59 Nagata, Y. et al. (1993) Isolation and characterization of Tn5-inducedmutants of Pseudomonas paucimobilis UT26 defective in g-hexa-chlorocyclohexane dehydrochlorinase (LinA). Biosci. Biotechnol.Biochem. 57, 703–709
60 Nagata, Y. et al. (1996) Isolation and characterization of Tn5-induced mutants of Sphingomonas paucimobilis defective in 2,5-dichlorohydroquinone degradation. Biosci. Biotechnol. Biochem. 60,689–691
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