Cloning of a novel dehalogenase from environmental DNA

Article abstract of DOI:10.1271/bbb.100027

Cloning of pce A, the gene of tetrachloroethene (PCE)- reductive dehalogenase, was undertaken from environmental DNA. Two genes were amplified using PCR primer deduced from pee A. Functional expression of these genes was unsuccessful in Escherichia coli, but PceA1 synthesized in vitro was enzymatically active. In recombinant E. coli, PceA1 formed a complex with host DnaK and caused filamentous growth.

Full text of DOI:10.1271/bbb.100027

Biosci. Biotechnol. Biochem., 74 (6), 1290–1292, 2010
Note
Cloning of a Novel Dehalogenase from Environmental DNA
y
Hisashi KIMOTO,1; Shin-ichiro SUYE,2 Hirokazu MAKISHIMA,2 Jun-ichirou ARAI,3
5
Sachiko YAMAGUCHI,3 Yutaka FUJII,4 Toshihito YOSHIOKA,1 and Akira TAKETO
1Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuokakenjojima, Eiheiji-cho, Yoshida-gun,
Fukui 910-1195, Japan
2Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui,
3-9-1 Bunkyo, Fukui 910-8507, Japan
3Daikin Industries, Ltd., Environmental Technology Laboratory, 1304 Kanaoka-cho, Kita-ku, Sakai,
Osaka 591-8511, Japan
4Department of Molecular Biology and Chemistry, Faculty of Medicine, University of Fukui,
23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan
5Department of Environmental and Biotechnological Frontier Engineering, Fukui University of Technology,
3-6-1 Gakuen, Fukui 910-8505, Japan
Received January 13, 2010; Accepted March 3, 2010; Online Publication, June 7, 2010
[doi:10.1271/bbb.100027]
Cloning of pceA, the gene of tetrachloroethene (PCE)-
reductive dehalogenase, was undertaken from environ-
mental DNA. Two genes were amplified using PCR
primer deduced from pceA. Functional expression of
these genes was unsuccessful in Escherichia coli, but
PceA1 synthesized in vitro was enzymatically active. In
recombinant E. coli, PceA1 formed a complex with host
DnaK and caused filamentous growth.
Using these primers and the Ex-Taq system (Takara Bio,
Shiga), gene fragments were amplified by PCR. Only
one sample among the 50 environmental DNA prepa-
rations yielded an amplified fragment. Subsequent
subcloning with a TOPO TA Cloning Kit (Invitrogen,
California, USA) revealed that this PCR product con-
tained two putative pceA genes (pceA1 and pceA2). The
full-length genes were obtained by Annealing Control
Primerꢀ technology, using DNA Walking SpeedUp
Premix Kit 2 (Seegene, Seoul, Korea). Amplification of
each full-length pce gene was confirmed by a single
PCR, using the environmental DNA template. The
amino acid sequences were deduced from the genes:
PceA1 was composed of 500 residues (56 kDa), and
PceA2 of 493 residues (55 kDa). A homology search
with the Basic Local Alignment Search Tool (BLAST)
(http://www.ncbi.nlm.nih.gov/BLAST/) showed that
both enzymes were highly homologous to PceA of
Sulfurospirillum (Dehalospirillum) multivorans. As
shown in Fig. 1, the homology levels were 93% (PceA1)
and 63% (PceA2). At the N-terminus, each enzyme
possessed a putative signal sequence containing the
twin-arginine motif (RRXFXK) as a Sec-independent
membrane translocation system. On the other hand, two
iron-sulfur cluster binding motifs (CXXCXXCXXXCP
and CXX[2{12]CXXCXXXCP) are present at each C-
terminus.1,2) In common with pceA of S. multivorans3)
and Desulfitobacterium sp. strain Y51,4) pceB, the gene
encoding the putative membrane anchor protein of
PceA, was located downstream of the pceA genes.
Next, the pceA1 and pceA2 genes deleted in the
putative signal sequences were subcloned individually in
pQE60 (Qiagen, Hilden, Germany) and introduced into
E. coli BL-21. By electroporation,5) six ampicillin-
resistant transformants were obtained, and each of them
was found to retain the intact insert and vector.
Expression was however detected only in two clones by
western blot analysis using anti-his-tag antibody, and the
Key words: dehalogenase; metagenome; tetrachloroe-
thene; tetrachloroethylene; DnaK
Owing to their durability under harsh chemical and
heat conditions, halogenated organic compounds are
used industrially, e.g., in the dry cleaning and washing
of semiconductor. Once released into the environment,
however, these compounds penetrate soils, accumulate
in ground water, and often cause long-range pollution.
Although physical and chemical methods (adsorption,
oxidation, etc.) are used in the treatment of soils and of
ground water contaminated with halogenated com-
pounds, the application of microbial cleaning capacity
in bioremediation is expected to prove a mild, safe
technique. From environmental DNA, we attempted to
clone the gene(s) of the enzyme involved in the
detoxification of halogenated organic compounds, espe-
cially tetrachloroethylene (PCE). Several reports have
appeared dealing with PCE reductive dehalogenase
(PceA), the enzyme that dehalogenates PCE. We
prepared environmental DNA from soil-containing
groundwater samples collected in 50 domestic industrial
zones. For DNA extraction, the ISOIL for Beads Beating
Kit (Nippon Gene, Toyama) was used. Synthetic primers
(50-GAYAARCCNATHGAYTTYGG-30 and 50-CCRT-
ANCCNARNGCRTCRTC-30) were designed, referring
to amino acid (nucleotide) sequences relatively con-
served in several known pceA genes (accession nos.
AF022812, AB194706, AY216592, and AY013367).
y
To whom correspondence should be addressed. Tel: +81-776-61-6000; Fax: +81-776-61-6011; E-mail: kimoto@fpu.ac.jp
Abbreviations: PCE, tetrachloroethene; PceA, PCE reductive dehalogenase; GFAT, L-glutamine-D-fructose-6-phosphate aminotransferase

Cloning and Expression of Dehalogenase in E. coli
1291
Fig. 1. Sequence Alignment of PCE Reductive Dehalogenases.
The chromosomal DNA sequence database was searched using BLASTP search algorithms. The amino acid sequence data were analyzed
using GENETYX computer software (Software Development, Tokyo). The homologous amino acid residues found in all three enzymes
(asterisks) and conserved substitutions (dots) are indicated. The deduced signal sequence containing the twin-arginine motif (RRXFXK) is
underlined. Two possible iron-sulfur cluster binding motifs, CXXCXXCXXXCP and CXX[2{12]CXXCXXXCP, are double-underlined. The
broken underlined amino acid sequences are conserved in a few known pceA genes. Based on these sequences, synthetic primers were designed
for cloning of the PCE reductive dehalogenase genes. The alignments were from the DDBJ/GenBank/EMBL accession numbers in parentheses,
as follows: PceA of Sulfurospirillum (Dehalospirillum) multivorans (AAC60788); PceA1 (AB537448); PceA2 (AB537449).
expressed enzyme protein formed an insoluble inclusion
body. The growth of the host cells, and hence yield of the
expressed product, decreased on IPTG treatment. In spite
of various experiments using vector pBAD, pCold and
different host strains, no PceA activities were detectable
in the transformant E. coli lysates.
In general, reductive dehalogenases are highly sensi-
tive to oxygen and refractory to purification, owing to
high content of cystein residue. Indeed the PceA1 and
PceA2 molecules had 15 and 12 cystein residues
respectively. Anaerobic cultures of E. coli and of yeast
cells, however, did not yield PceA1 activities.
In consideration of these difficulties, PceA1 was
synthesized in vitro using the WakoPURE system
(Wako Pure Chemical Industries, Osaka).6) After re-
placement of air phase with N2 gas, the reaction mixture
was incubated at 37 ꢀC for 1 h. The resuling enzyme, the
concentration of which was below the detection limit
on SDS–PAGE, was assayed by the method of Neumann
et al.7) In brief, the synthetic enzyme was mixed with

1292
kDa
H. KIMOTO et al.
1
2
3
A
250
150
100
75
50
37
DnaK
GFAT
PceA1
25
20
Prolyl isomerase
15
10
B
PceA1
(degradattion product)
Fig. 2. Direct Interaction between His-Tag Fused PceA and DnaK in
Vivo (His-tagged protein pull-down assay).
A culture of E. coli BL21 harboring recombinant plasmid (pCold
harboring pceA1) or mock plasmid (pCold) was induced with 1 mM
IPTG for 24 h at 15 ꢀC, and the His tag-fused recombinant PceA1 or
vector control was extracted, using BugBusterꢀ protein extraction
reagent (Novagen, Madison, WI) containing 1.0 mM PMSF and
0.001 mM pepstatin. BugBusterꢀ extract was loaded on a Ni-NTA
Superflow Cartridge (Qiagen, Hilden, Germany), and the His tag-
fused PceA1 that stuck to the cartrige (lane 2) and the control (lane
3) were recovered with 250 mM imidazole. The eluates were
subjected to 15% SDS–PAGE and stained with CBB R-250 dye.
The proteins in the gel were transferred to a PVDF membrane, and
the bands were cut off and analyzed with a PE Applied Biosystems
model 491 Procise protein sequencer. GFAT stands for L-glutamine-
D-fructose-6-phosphate aminotransferase.
Fig. 3. Filamentation of the E. coli pceA Transformant.
Cells of strain XL1-Blue transformed with pPceA (pBAD bearing
the pceA insert) (A) or pBAD (the insert-free control, B) were grown
at 37 ꢀC for 6 h in the presence of 0.2% L-arabinose, and were
subjected to phase-contrast microscopy.
In spite of analysis of the enzymes and genes involved
in dehalogenative respiration, efficient expression of
PceA is still difficult, and establishment of gene
manipulation system is urgently required. Furthermore,
most dehalogenative bacteria are refractory to pure
culture, and the development of a culture system is also
essential. The PCR primers used in this study, designed
for the cloning of pceA, are probably applicable in
searching for new allied genes or dehalogenative
bacteria, and in the monitoring of microbes in bioreme-
diation.
PCE in Tris-pyruvate buffer, pH 7.3, containing methyl
viologen, titanium citrate, DTT, and BSA, followed by
incubation at 30 ꢀC. Care was taken to minimize
exposure to oxygen. After incubation for 1–4 h, the
mixture was heated at 65 ꢀC for 15 min, and the head-
space gas, containing evaporated PCE and TCE, was
subjected to GC/MS analysis. The protein synthesized
in vitro dehalogenated PCE into trichloroethylene
(TCE), at a rate of 2.30 mmole/h ml of reaction mixture.
Except when determined immediately after synthesis,
dehalogenase activity was undemonstrable, indicating
high oxygensensitivity.
As mentioned above, the PceA of S. multivorans
(D. multivorans) has 93% homology with PceA1, and
the recombinant protein expressed in E. coli is similarly
deficient in dehalogenase activity.3) On the other hand,
PceA did not exhibit toxicity against E. coli, and the
amount of the expressed product was not small. The
toxicity of PceA1 was unrelated to dehalogenase activity
and its signal peptide.
A search for the host protein interacting with PceA1
was performed, using pull-down assay.8) The PceA1
molecule formed a complex with the DnaK (Hsp70)9)
protein (Fig. 2). Through interaction with DnaK, a
molecular chaperon involved in DNA replication,
PceA1 probably shows toxicity against host E. coli.
In accordance with the molecular interaction, E. coli
cells harboring the pceA1 plasmid underwent filament
formation (Fig. 3), one of the properties of dnaK
mutants.10) Like dnaK strains,10) the pceA transformant
grew slowly, and easily lost the pPceA plasmid.
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