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Journal of Bacteriology, August 1998, p. 4140-4145, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Tetrachloroethene Dehalogenase from Dehalospirillum
multivorans: Cloning, Sequencing of the Encoding Genes, and
Expression of the pceA Gene in Escherichia
coli
Anke
Neumann,
Gert
Wohlfarth, and
Gabriele
Diekert*
Institut für Mikrobiologie der
Universität Stuttgart, Stuttgart, Germany
Received 12 November 1997/Accepted 5 June 1998
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ABSTRACT |
The genes encoding tetrachloroethene reductive dehalogenase, a
corrinoid-Fe/S protein, of Dehalospirillum multivorans were cloned and sequenced. The pceA gene is upstream of
pceB and overlaps it by 4 bp. The presence of a 
70-like
promoter sequence upstream of pceA and of a 
-independent
terminator downstream of pceB indicated that both genes are
cotranscribed. This assumption is supported by reverse transcriptase
PCR data. The pceA and pceB genes encode putative 501- and 74-amino-acid proteins, respectively, with
calculated molecular masses of 55,887 and 8,354 Da, respectively.
Four peptides obtained after trypsin treatment of
tetrachloroethene (PCE) dehalogenase were found in the deduced
amino acid sequence of pceA. The N-terminal amino acid
sequence of the PCE dehalogenase isolated from D. multivorans was found 30 amino acids downstream of the N
terminus of the deduced pceA product. The pceA
gene contained a nucleotide stretch highly similar to binding
motifs for two Fe4S4 clusters or for one
Fe4S4 cluster and one
Fe3S4 cluster. A consensus sequence for the
binding of a corrinoid was not found in pceA. No
significant similarities to genes in the databases were
detected in sequence comparisons. The pceB gene contained
two membrane-spanning helices as indicated by two hydrophobic stretches
in the hydropathic plot. Sequence comparisons of pceB
revealed no sequence similarities to genes present in the databases.
Only in the presence of pUBS 520 supplying the recombinant
bacteria with high levels of the rare Escherichia coli
tRNA4Arg was pceA expressed, albeit
nonfunctionally, in recombinant E. coli BL21 (DE3).
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INTRODUCTION |
Dehalospirillum
multivorans is a strictly anaerobic, gram-negative bacterium,
which is able to grow with tetrachloroethene (PCE) as the terminal
electron acceptor for the oxidation of different electron donors
(14, 15, 19). The bacterium is able to grow at the expense
of H2 and PCE. Since H2 oxidation cannot be
coupled to ATP synthesis via substrate level phosphorylation, the
reductive dechlorination of PCE has to be the energy-generating
process, probably via a chemiosmotic mechanism. Therefore, this
process was referred to as PCE respiration (25).
The key enzyme of the reductive part of catabolism, PCE reductive
dehalogenase, mediates in vitro the reductive dechlorination of PCE via
trichloroethene to cis-1,2-dichloroethene with
reduced methyl viologen as the electron donor. PCE dehalogenase was
purified from the cytoplasmic fraction of D. multivorans cells (16). The enzyme contains a corrinoid
as well as about eight iron atoms and eight acid-labile sulfur atoms.
The corrinoid is involved in this reaction presumably as a redox-active
prosthetic group, as deduced from the finding that it has to be reduced
to cob(I)alamin prior to the nucleophilic attack on the carbon of PCE
(13, 25). This reaction represents a completely new
type of biochemical reaction. Here we describe the cloning and
sequencing of the genes of the PCE dehalogenase for further
characterization of the enzyme and for comparison with the genes of
other proteins.
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MATERIALS AND METHODS |
Determination of amino acid sequences of PCE dehalogenase.
D. multivorans was grown as previously described
(14). PCE dehalogenase was isolated from the organism as
described elsewhere (16). The protein was treated with
trypsin (22). The peptides obtained were separated by
high-pressure liquid chromatography with a Grom-Sil 300 octyldecyl
silane column, and the N-terminal amino acid sequences of four peptides
and of PCE dehalogenase were determined by H. Weber at the
Fraunhofer-Institut für Grenzflächen und
Bioverfahrenstechnik (Stuttgart, Germany) or V. Nödinger at the
Institut für Technische Biochemie (University of Stuttgart, Stuttgart, Germany).
Cloning of pceA.
The isolation of DNA from
D. multivorans, restriction, DNA ligation, and other
standard techniques were performed as described elsewhere
(2). Plasmid DNA for cloning and sequencing was prepared with the Flexi Prep kit (Pharmacia, Freiburg, Germany). Properties of
plasmids used in this study are summarized in Table
1.
A homologous probe for
pceA was generated by PCR with
genomic DNA from
D. multivorans as the template. The
oligonucleotides
(GGI GAG GTI AAG CCI TGG TT and GTC CCA IAC YTC IGT
DAT RTT) were
derived from the internal peptides GEVKPWFLXAYD and
NITEVWDGK
(Fig.
1). PCR mixtures (50 µl) for the amplification of
genomic
DNA contained 50 pmol of each primer, 0.1 µg of chromosomal
template
DNA, a 0.1 mM concentration of each deoxynucleotide
triphosphate,
Goldstar DNA polymerase reaction buffer, and 1 mM
MgCl
2. The PCR
program started with initial denaturing (3 min, 96°C). The addition
of 0.5 U of Goldstar DNA polymerase
(Eurogentech, Cologne, Germany)
was followed by 29 cycles of
polymerization (1 min, 45°C; 1.5
min, 72°C; 0.5 min, 95°C) and a
final cycle with prolonged polymerization
time (15 min, 72°C). A
1.2-kb fragment was amplified and cloned
into a T-tailed vector
(
11) prepared from pBluescript II SK+
(Stratagene,
Heidelberg, Germany). The resulting plasmid, named
pW3, was partially
sequenced. The identity of the fragment was
confirmed by comparison of
the deduced amino acid sequence with
the peptide sequences of PCE
dehalogenase.
Genomic DNA was digested with several restriction endonucleases. The
DNA fragments generated were separated by agarose gel
electrophoresis,
transferred to a nylon membrane by the capillary
transfer method
(
21), and hybridized at 68°C with the 1.2-kb
PCR product
labeled with digoxigenin (DIG) by using the DIG DNA
labeling and
detection kit (nonradioactive) as indicated by the
supplier
(Boehringer, Mannheim, Germany). Genomic
EcoRI fragments
were isolated from agarose gels (Gene Clean II; Bio 101, La Jolla,
Calif.), ligated into pBluescript II SK+, and transformed into
E. coli DH5
cells (
9). Positive clones were identified
by
Southern hybridization with the DIG-labeled 1.2-kb PCR product
by
using the DIG DNA detection kit (nonradioactive). One clone,
named
pY179, containing a 6-kb
EcoRI fragment was used for further
analyses.
DNA sequencing and analyses.
The nucleotide sequence of the
6-kb EcoRI fragment was determined by sequencing pY179 and
subclones of pY179. For the sequencing reactions, an Applied Biosystems
Prizm kit (Weiterstadt, Germany) was used, with subsequent
electrophoresis and analyses in an Applied Biosystems A373 sequencer.
Oligonucleotides (about 30 bases) were used for sequencing the
remaining gaps. Both strands were independently and completely
sequenced.
Computer-assisted DNA and protein sequence analyses, alignments, and
hydropathic plots were performed with the software package
PC/Gene
(IntelliGenetics Inc., Geneva, Switzerland). Database
searches were
performed with BLAST (
1).
RNA isolation and analysis.
Total RNA was isolated from
freshly grown cells of D. multivorans grown on pyruvate
and fumarate or of Escherichia coli BL21 (DE3)/pLysS/pPCEA
grown on Luria broth plus 100 µM ampicillin, induced with 1 mM IPTG
(isopropyl-1-
-D-thiogalactopyranoside) by using the
RNeasy Mini Kit (Qiagen, Hilden, Germany) as described by the supplier.
Remaining DNA was digested with DNase I (RNase free; Pharmacia) for 10 min at 37°C in an assay mixture (10 µl) containing 1 to 2 µg of
RNA, 3.25 U of DNase I, 3 mM MgCl2, 2 µl of 5×
first-strand buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM
MgCl2; Gibco BRL, Eggenstein, Germany), and 20 U of RNaseOUT (Gibco BRL). Subsequently, the mixture was incubated for 10 min at 70°C to denature the DNase I. cDNA synthesis was done with
SuperScript II RNase H
reverse transcriptase (RT) (Gibco
BRL). The RT-PCR assay mixture (20 µl) contained 9 µl of DNA-free
RNA, 20 U of RNaseOUT, 2 µl of 5× first-strand buffer, 10 mM
deoxynucleoside triphosphate, 2 pmol of primer, and 200 U of RT.
The Cy5-labeled primer (Pharmacia) for the primer extension method
(
6) was constructed complementary to the first 30 bases
of
pceA (TGA GAG TTC AGG CTT TTT TTT CTT TTC CAT). The length
of synthesized cDNA was analyzed with an ALFex-press DNA sequencer
(Pharmacia).
RT-PCR experiments were performed with RT-PCR beads (Pharmacia). Five
to ten microliters of DNase-digested RNA was used as
the template. The
primers for cDNA and PCR are given in the Results
section. The
procedure was performed according to the provider's
instructions.
Expression of the PCE dehalogenase.
The coding regions of
pceA and pceAB were amplified from pY179 by PCR
as described above by introducing heterologous NcoI and
BamHI sites. The primers for pceA were ATA GAC
CAT GGA AAA GAA AAA AAA GCC TGA ACT CTC and GCA AGG ATC CTC ATG ATT TTT
TAA CCC TAT CCT TTC TAA AGC; the primers for pceAB were ATA
GAC CAT GGA AAA GAA AAA AAA GCC TGA ACT CTC and AGC TGG ATC CTT AAC GCT TAA GCT TTT CCC ATA AAA TAT ATG; the primers for pceA'
(pceA without the leader sequence; Fig. 1) were AAC AAC CAT
GGG TGT ACC AGG TGC AAA TGC and GCA AGG ATC CTC ATG ATT TTT TAA CCC TAT
CCT TTC TAA AGC. The PCR products were ligated into expression vector pET 11d between the NcoI and BamHI sites, and the
vector was transformed into E. coli DH5. Sequencing (300 bp from each side) revealed the correct position of the inserts in
pPCEA, pPCEA', and pPCEAB. Plasmids pPCEA, pPCEA', pPCEAB, and pET 11d
were transformed into E. coli BL21 (DE3)/pLysS (Stratagene)
or E. coli BL21 (DE3)/pUBS 520. Freshly transformed E. coli cells were used to inoculate a 50-ml overnight culture (Luria
broth plus 100 µg of ampicillin per ml at 30°C). One liter of the
same medium was inoculated with this culture and induced with 0.4 mM
IPTG at an optical density at 600 nm (OD600) of between 0.5 and 0.7. Samples (250 ml) were taken at 0, 1.5, 3, and 4 h after
induction, centrifuged for 15 min at 6,000 × g,
suspended in 1 ml of 0.1 mM Tris-HCl (pH 7.5) per 0.5 OD600
unit, and disrupted with a French press. The crude extracts were
analyzed by sodium dodecyl sulfate-12% polyacrylamide gel
electrophoresis (SDS-12% PAGE).
D. multivorans extracts for supplementation of the
growth medium were prepared by suspending 30 g of cells (wet
weight; grown
on pyruvate and fumarate) in 70 ml of 0.1 M potassium
phosphate
buffer, pH 7.5. The cells were disrupted with a French press.
The crude extract was autoclaved and centrifuged for 20 min at
8,000 ×
g. One liter of
E. coli minimal
medium (
8) was supplemented
with 25 ml of the supernatant.
Nucleotide sequence accession number.
The sequences
described in the present manuscript have been deposited in GenBank
under accession no. AF022812.
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RESULTS |
Cloning and sequencing the PCE dehalogenase gene.
PCE
dehalogenase from D. multivorans was purified from the
cytoplasmic fraction of pyruvate- and fumarate-grown cells as described elsewhere (16). The N terminus of the purified protein was
sequenced. It started with the amino acid glycine, indicating a
posttranslational processing of the protein. In addition, the PCE
dehalogenase was digested with trypsin and the amino acid sequences of
four of the resulting peptides were determined (Fig.
1). Two degenerated oligonucleotides were
derived from the amino acid sequences of peptides 1 and 4 (Fig. 1). A
1.2-kb fragment was amplified by PCR with genomic DNA of D. multivorans as the template. The PCR product was cloned in
pBluescript, and resulting plasmid pW3 was partially sequenced. All
four peptides were found in the deduced amino acid sequence encoded by
the 1.2-kb fragment. Given the apparent molecular mass of 58 kDa for
the PCE dehalogenase (16), it was calculated that the PCR
product contained about 70% of pceA (PCE dehalogenase
gene). A gene probe labeled with DIG was prepared with the PCR product
as the template.
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FIG. 1.
DNA sequence of the operon containing the genes for the
PCE dehalogenase from D. multivorans and the deduced
amino acid sequence. Putative E. coli 70 promoter
sequences ( 35 and 10 regions) are overlined. A possible terminator
sequence is underlined with broken arrows, and ribosome binding sites
are marked with asterisks. The leader peptide is in boldface. Solid
lines below the deduced amino acid sequence encoded by pceA
indicate sequences also determined by Edman degradation.
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Genomic DNA from
D. multivorans was digested with
several restriction endonucleases, and the fragments were separated by
agarose
gel electrophoresis. Southern blot analysis of the genomic DNA
fragments with the gene probe identified an
EcoRI fragment
of
about 6 kb containing at least part of the
pceA gene.
Thus, genomic
EcoRI fragments of 5.5 to 6.5 kb were isolated
from agarose gels,
ligated into the pBluescript vector, and transformed
into
E. coli DH5
. Three of 200 clones hybridized with the
gene probe. The
plasmid of one of the positive clones, pY179, contained
an
EcoRI
insert of about 6 kb and was used for double-strand
sequencing.
Sequence analysis of the PCE dehalogenase gene.
An open
reading frame (ORF) coding for a protein (product of pceA)
which harbors the N terminus and all four internal peptides of PCE
dehalogenase was found on the 6-kb EcoRI fragment (Fig. 1).
The sequence of the product of this ORF started 30 amino acids (corresponding to 90 bp of the ORF) upstream of the N terminus of PCE
dehalogenase isolated from D. multivorans. In the
N-terminal part of the deduced protein encoded by pceA, a
putative signal sequence, RRXFXK, followed by a stretch of hydrophobic
amino acids was detected (5) (Fig.
2). These findings support the assumption of a processing of the protein. During the first steps of purification of PCE dehalogenase, a protein with an apparent molecular mass of 61 kDa was copurified (Fig. 6, lane 6, band B). The N-terminal sequence of
this protein was identical to the N-terminal amino acid sequence of the
protein deduced from pceA, indicating that this protein was
the unprocessed PCE dehalogenase. The molecular masses of the deduced
501-amino-acid protein (nonprocessed) and of the truncated
471-amino-acid protein were calculated to be 55,887 Da and 52,674 Da,
respectively. Taking into consideration the fact that PCE dehalogenase
contains a corrinoid and about eight iron and eight acid-labile sulfur
atoms (16), the calculated size of the truncated holoenzyme
(about 55 kDa) is in accordance with the apparent molecular mass of the
native PCE dehalogenase determined by gel filtration (58 kDa). In the
amino acid sequence deduced from pceA, consensus sequences
similar to that for two Fe4S4 clusters
(CXXCXXCXXXCP; 7) were identified from amino acids 365 to 377 and 420 to 428 (Fig. 1). The only difference from the consensus sequence is a
glycine instead of a cysteine at amino acid position 417. A consensus
sequence for the binding of a corrinoid (DXHXXG; 12) could not be
detected. No other significant similarities to genes in the databases
were found in sequence comparisons. In addition, no significant
similarities were revealed by amino acid sequence comparisons of the
pceA gene product with vitamin B12 and
coenzyme B12-binding proteins performed with the software package PC/Gene.
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FIG. 2.
Alignment of the N terminus encoded by pceA
from D. multivorans with signal peptide sequences of
the small subunits of hydrogenases from Alcaligenes
eutrophus and E. coli, dimethyl sulfoxide
reductase from Rhodobacter sphaeroides, and the large
subunit of formate dehydrogenase from W. succinogenes. The
data were taken from reference 5. The sequences have
been aligned relative to the consensus sequence RRXFXK (in boldface).
Cleavage sites are marked by arrows.
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The start codon of a further ORF (
pceB) overlaps by four
bases the C terminus-encoding region of
pceA (Fig.
1). The
74-amino-acid
protein encoded by this gene (225 bp) has a
calculated molecular
mass of 8,354 Da. Two hydrophobic regions were
detected in the
hydropathic plot of this protein, indicating the
presence of two
membrane-spanning helices (Fig.
3). Cysteine and histidine residues
were
not detected in the deduced amino acid sequence of
pceB.
In
addition, sequence comparisons revealed no significant similarities
to
genes present in the databases.
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FIG. 3.
Hydropathic plot of the predicted amino acid sequence of
pceB. The plot was obtained by using the software package
PC/Gene (IntelliGenetics Inc.).
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Each of the start codons of
pceA and
pceB is
preceded by a putative ribosome binding site (Fig.
1). As shown in
Fig.
1, there
are two stretches resembling the
10 and
35 regions of
an
E. coli 70 promoter, indicating a transcription start
between positions
3160 and 3170. The primer extension method, with
total RNA isolated
from
D. multivorans as the template,
was used to determine that
the transcription start site of the PCE
dehalogenase was at approximately
position 3180 (Fig.
4). In addition, RT-PCR with two
different
oligonucleotide pairs (pair I: positions 3162 to 3194 and 3501
to 3476; pair II: positions 3090 to 3117 and 3501 to
3476) was
conducted with total RNA from
D. multivorans.
Only with pair I
was a PCR product (size, 339 bp) obtained, indicating
a transcription
start between positions 3117 and 3161. Downstream of
the
pceB stop codon, an inverted repeat followed by a
poly(T) stretch (Fig.
1), which possibly acts as a
-independent
terminator, was detected.
RT-PCR with an oligonucleotide pair
(positions 4660 to 4682 and
4992 to 4967) revealed a 335-bp PCR
product, indicating cotranscription
of
pceA and
pceB (data not shown).
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FIG. 4.
Mapping of the pce transcription start site
by the primer extension method. The primer for cDNA synthesis and
sequencing of Y179 was complementary to the nucleotides at positions
3301 to 3272. Curves A, C, G, and T show the sequencing of Y179. The
numbers under the bases correspond to the numbering in Fig. 1. For
further details, see Materials and Methods.
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Genes upstream of the pceAB genes.
Upstream of the
pceAB genes, one ORF (ORF1) with no significant sequence
similarities to genes in the databases was detected. Further upstream
of ORF1, three ORFs were identified as genes encoding enzymes of the
riboflavin biosynthesis pathway. The first ORF (ribA)
encodes the C-terminal part of a protein with the greatest sequence similarity to GTP cyclohydrolase II of Helicobacter
pylori (81 of 141 amino acids identical). The two other ORFs
encode proteins with sequence similarities to the
3,4-dihydroxy-2-butanone-4-phosphate synthase (ribB) of
Photobacterium phosphoreum (115 of 356 amino acids
identical) and to the beta subunit of the riboflavin synthase (ribH) of E. coli (81 of 156 amino acids
identical). Each of the start codons of the ORFs is preceded by a
putative ribosome binding site. An overview of the DNA region
comprising the genes for PCE dehalogenase is given in Fig.
5.
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FIG. 5.
Physical map of the DNA region comprising the genes for
PCE dehalogenase. For further details, see the text.
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Expression of the PCE dehalogenase genes in E. coli BL21.
The pceA gene was amplified by PCR
with pY179 as the template. The PCR product was cloned in pET 11d
downstream of an IPGT-inducible T7/lac promoter and
transformed into E. coli BL21 (DE3)/pLysS. After
induction of the bacteria with 0.4 mM IPTG, neither PCE dehalogenase
activity nor a protein of the molecular size of PCE dehalogenase as
determined by SDS-PAGE could be detected in crude extracts. RT-PCR of
RNA isolated from IPTG-induced bacteria revealed the expected DNA
fragments indicating the transcription of the gene (data not shown).
Analysis of the codon usage of the
D. multivorans gene
in pY179 revealed that the triplet AGA coding for arginine is used
much
more frequently than in
E. coli (data not shown).
Moreover,
two AGA tandems are present in
pceA (positions
3302 to 3307 and
3776 to 3781 in Fig.
1). In
E. coli,
AGA codons are translated
by the rare tRNA
4Arg
encoded by
argU (
18).
The plasmid pUBS 520 containing
argU was transformed in
E. coli BL21 (DE3) harboring
pceA,
pceA', or
pceAB (see Table
1).
After induction of
the bacteria with 0.4 mM IPTG, proteins with
molecular masses of
61 ± 1 kDa (
pceA product) and 57 ± 1 kDa
(
pceA'
product) were expressed in crude extracts of the
respective recombinant
bacteria as determined by SDS-PAGE (shown for
pceA in Fig.
6).
PCE
dehalogenase activity could not be detected in crude extracts
of
recombinant
E. coli. Growth of the recombinant bacteria
under
anaerobic conditions or the addition of vitamin B
12
or of autoclaved
crude extract of
D. multivorans to the
medium did not result in
functional expression of PCE dehalogenase.
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FIG. 6.
Expression of pceA from D. multivorans in E. coli BL21 (DE3) as analyzed by
SDS-PAGE. Strains of E. coli BL21 (DE3) harboring
different plasmids were induced by IPTG. Cell extracts were analyzed by
SDS-12% PAGE and subsequently stained with Coomassie brilliant blue
G-250. Lane 1, molecular mass markers (molecular mass is given in
kilodaltons); lane 2, E. coli/pLysS/pET 11d; lane 3, E. coli/pUBS 520/pET 11d; lane 4, E. coli/pLysS/pPCEA; lane 5, E. coli/pUBS 520/pPCEA;
lane 6, PCE dehalogenase enriched from D. multivorans
(fraction eluted from the phenyl Superose column; reference
16); lane 7, same as lane 5; lane 8, same as lane 1. A, pceA gene product; B and C, unprocessed and processed
D. multivorans PCE dehalogenase, respectively, as
confirmed by N-terminal amino acid sequence analysis.
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DISCUSSION |
We show here for the first time cloning, sequencing, and
expression of a PCE reductive dehalogenase from gram-negative, strictly anaerobic D. multivorans. Evidence for the presence of
two ORFs, designated pceA and pceB, on the PCE
dehalogenase operon is presented. From the finding that a putative
E. coli 70 promoter region precedes pceA
and a -independent terminator structure follows the stop codon of
pceB, it was concluded that the two genes form one operon. This assumption was further supported by RT-PCR experiments indicating that both ORFs are cotranscribed. ORF pceA encodes the PCE
dehalogenating protein. The finding that pceB encodes a
highly hydrophobic protein with two transmembrane helices suggests that
the gene product might be a membrane-anchoring subunit for the
attachment of the pceA gene product to the cytoplasmic
membrane. This is feasible, since the PCE dehalogenase is involved in a
respiratory process (13, 17). No significant similarities of
the pceB product to other proteins was found in sequence
comparisons. Since the amino acids histidine and cysteine were lacking
in the pceB gene product, a binding of heme to the protein
as in cytochromes or of Fe/S clusters is not feasible, indicating that
this "subunit" is probably not involved in the electron transport
chain.
In the upstream region of pceA, three ORFs
(ribABH) were identified as encoding putative enzymes of
riboflavin biosynthesis; one ORF could not be identified. No ORF could
be detected in the 0.9-kb downstream region of pceB.
Putative ribosome binding sites were detected upstream of the start
codon of each gene, indicating that the genes could be expressed in
D. multivorans.
From the finding that the N terminus of PCE dehalogenase was found
downstream of the N terminus of the deduced pceA protein, it
is concluded that the protein was modified by truncation of the first
30 amino acids in D. multivorans. The modification
signal was probably the peptide RRXFXK followed by a hydrphobic
stretch, which was mainly reported for periplasmic,
cofactor-binding proteins (5). This is surprising, since PCE
dehalogenase was recovered exclusively in the cytoplasmic fraction of
D. multivorans. The only other protein containing this
leader sequence and facing the cytoplasmic side of the membrane is the
dimethyl sulfoxide reductase of E. coli. This enzyme
was reported to contain a hydrophobic subunit, which obviously hampers
the catalytically active subunits from being excreted into the
periplasm (24). Hence, it is feasible that the product of
pceB serves a similar function for PCE dehalogenase. Usually, the cleavage sites for these and the Sec signal peptides are
preceded by two small amino acids at positions 1 and 3
(23). In the pceA gene product, this site is
preceded by phenylalanine at position 1 (Fig. 2).
The deduced amino acid sequence of corrinoid-iron/sulfur protein PCE
dehalogenase exhibits no significant similarities to those of other
proteins, including other cobalamin-containing enzymes. In addition,
the cobalamin-binding site DXHXXG described for, e.g., the
cobalamin-dependent methionine synthase (3) as well as for
several adenosylcobalamin-containing mutases (4, 12), could
not be detected. Since there are corrinoid-containing enzymes, which
also lack this binding site, e.g., the corrinoid-iron/sulfur protein of
Clostridium thermoaceticum (10), the corrinoid is probably noncovalently bound to the PCE dehalogenase. Evidence based on
electron paramagnetic resonance (EPR) data is available for the PCE
dehalogenase of Dehalobacter restrictus and shows that the
cobalamin in this enzyme is a base-off corrinoid (20), suggesting that the cobalt is not coordinated with a histidine of the
apoenzyme. Assuming a similar structure for the enzyme of D. multivorans, the lack of a cobalamin binding site is not surprising.
A consensus sequence very similar to that for
Fe8S8 ferredoxins (two CXXCXXCXXXCP stretches;
7) was detected in the deduced amino acid sequence encoded by
pceA. However, the first cysteine of the second stretch is
replaced by glycine. There are three possible explanations for this
finding: (i) one iron atom of an Fe4S4 cluster
is not bound to the sulfur of cysteine; (ii) one of the clusters
contains only three Fe atoms; (iii) one iron atom of an
Fe4S4 cluster is bound to the sulfur atom of
cysteine, which is not located in the cluster. EPR experiments
performed with the PCE dehalogenase of D. restrictus
indicated the presence of 8 Fe atoms (20). Until now,
the absence of cysteine at the first position of the second stretch has
never been reported for Fe/S proteins (examples are given in Fig.
7). Database research revealed a
consensus of both stretches with only three other sequences, probably
those of unknown proteins (Fig. 7). The elucidation of the structure of
the iron-sulfur clusters will have to await EPR studies on the PCE
dehalogenase of D. multivorans.
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FIG. 7.
Alignment of the putative Fe/S-binding region of the PCE
dehalogenase (pceA product) from D. multivorans with the corresponding regions of other Fe/S proteins.
The iron-binding cysteines and the prolines are in boldface. The arrow
marks the missing cysteine in the second stretch of the Fe/S-binding
region of the PCE dehalogenase and of three "hypothetical proteins"
(HP) in comparison to sequences of Fe8S8 and
Fe7S8 ferredoxins (Fd). B. stearothermophilus, Bacillus stearothermophilus;
S. sp. strain PCC6803, Synechocystis sp. strain
PCC6803; T. thermosaccharolyticum,
Thermoanaerobacterium thermosaccharolyticum; C. pasteurianum, Clostridium pasteurianum; T. acidophilum; Thermoplasma acidophilum;
D. africanus, Desulfovibrio africanus;
B. schlegelii, Bacillus schlegelii; R. capsulatus, Rhodobacter capsulatus.
|
|
The high PCE concentrations detected in groundwater and soils of
contaminated sites is due to extensive use of this compound as a
solvent during the last 50 years. It is surprising that PCE dehalogenase has no significant homology to other proteins known so
far, although it has to be assumed that the enzyme was developed from ancestors within this short period of time. The evolutionary origin of the enzyme remains to be unraveled.
The codon usage of D. multivorans differs from that of
E. coli especially with respect to the arginine tRNA
codons. In the genes of D. multivorans known so far,
the frequency of the codon AGA is about the same as in closely related
Wolinella succinogenes and about 10 times higher than in
E. coli. Since AGA tandems, which often result in the
formation of truncated gene products (18), are present in
pceA, it was not surprising that the gene was only expressed
in E. coli in the presence of pUBS 520, which supplied
the recombinant bacteria with high levels of the rare E. coli tRNA4Arg for the AGA codon. Crude extracts of
the recombinant E. coli did not exhibit
PCE-dehalogenating activity. The protein expressed in E. coli has an apparent molecular mass of 61 kDa, which was about 4 kDa larger than that of the PCE dehalogenase isolated from
D. multivorans. Therefore, it is feasible that the
61-kDa gene product is an unprocessed PCE dehalogenase. This might be one of the reasons why the 61-kDa PCE dehalogenase was not
enzymatically active. Although the apparent signal peptide RRXFXK
should be processed by E. coli under anaerobic
conditions (17), no modification of the pceA
protein occurred. A possible explanation may be that no cofactors were
bound to the dehalogenase in E. coli, while only
proteins containing bound cofactors are processed in the signal
peptide-carrying proteins (5). We also tried to express the
pceA' gene encoding the processed protein in E. coli. Although the protein was expressed, no PCE dehalogenase
activity could be detected. Possibly the corrinoid and/or the Fe/S
clusters have not been incorporated in the protein. Since
E. coli is not able to synthesize corrinoids, vitamin
B12 was added to the growth medium of the recombinant
bacteria. The finding that no activity could be detected in the crude
extracts of the supplemented bacteria may be due to the possibility
that D. multivorans uses corrinoid derivatives other
than vitamin B12. To test this hypothesis, autoclaved crude extracts of D. multivorans were added to the
growth medium supplying the recombinant E. coli with
D. multivorans corrinoids. Supplementation of the
medium with D. multivorans corrinoids did not
result in functional expression. It is feasible that the cobalamins were not taken up by E. coli under the experimental
conditions applied.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the Deutsche
Forschungsgemeinschaft, the BMBFT, and the Fonds der Chemischen
Industrie.
We gratefully acknowledge helpful discussions with J. Altenbuchner and
R. Mattes (Stuttgart, Germany). The plasmid pUBS 520 was kindly
provided by R. Mattes (Stuttgart, Germany).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Allmandring 31, D-70550 Stuttgart, Germany.
Phone: 49-(0)711-6855483. Fax: 49-(0)711-6855725. E-mail:
imbgd{at}po.uni-stuttgart.de.
| |
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Journal of Bacteriology, August 1998, p. 4140-4145, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
