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(Received for publication, September 14, 1995; and in revised form, December 5, 1995) From the
The chlorobenzene degradation pathway of Pseudomonas sp. strain P51 is an evolutionary novelty. The first enzymes of
the pathway, the chlorobenzene dioxygenase and the cis-chlorobenzene dihydrodiol dehydrogenase, are encoded on a
plasmid-located transposon Tn5280. Chlorobenzene dioxygenase
is a four-protein complex, formed by the gene products of tcbAa for the large subunit of the terminal oxygenase, tcbAb for the small subunit, tcbAc for the ferredoxin, and tcbAd for the NADH reductase. Directly downstream of tcbAd is the gene for the cis-chlorobenzene dihydrodiol
dehydrogenase, tcbB. Homology comparisons indicated that these
genes and gene products are most closely related to those for toluene (todC1C2BAD) and benzene degradation (bedC1C2BA and bnzABCD) and distantly to those for biphenyl, naphthalene, and
benzoate degradation. Similar to the tod-encoded enzymes,
chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol
dehydrogenase were capable of oxidizing 1,2-dichlorobenzene, toluene,
naphthalene, and biphenyl, but not benzoate, to the corresponding
dihydrodiol and dihydroxy intermediates. These data strongly suggest
that the chlorobenzene dioxygenase and dehydrogenase originated from a
toluene or benzene degradation pathway, probably by horizontal gene
transfer. This evolutionary event left its traces as short gene
fragments directly outside the tcbAB coding regions.
Bacteria that are able to use mono- or dicyclic aromatic
compounds as their sole source of carbon and energy under aerobic
growth conditions are present ubiquitously in the
environment(1, 2) . Considering the potential use and
importance of such bacteria to help to remove many man-made polluting
compounds, it is necessary to study the genetic and biochemical
variations found among these types of bacteria and to investigate their
evolutionary development(3) . Only then can the limitations of
existing metabolic pathways be understood and concepts be developed to
select or engineer novel pathways(4, 5) . Of special
interest will be to obtain degradation of highly recalcitrant
chlorosubstituted aromatic compounds, such as chlorinated benzenes and
biphenyls(6) . A very important enzyme complex in the
aerobic degradation of many aromatic compounds is the multicomponent
aromatic ring dioxygenase(7, 8) . Aromatic ring
dioxygenases, such as benzoate dioxygenase(9) , toluate
dioxygenase(10) , naphthalene(11) , biphenyl,
toluene(12) , or benzene dioxygenases(13) , are enzyme
complexes with three- or four protein subunits. This complex catalyzes
a redox reaction in which molecular oxygen is incorporated in the
aromatic ring at the expense of the oxidation of
NADH(7, 8, 12) . The resulting intermediate
is a cis-dihydrodiol derivative of the aromatic ring
structure. A dihydrodiol dehydrogenase then catalyzes the (formal)
oxidation of the dihydrodiol to a dihydroxy derivative and regenerates
the reduced NADH. The components of the aromatic ring dioxygenase
consist of different electron transport proteins (a ferredoxin and a
reductase, or a combined ferredoxin-NADH reductase) and the terminal
oxygenase (also called hydroxylase component or iron sulfur protein),
which is thought to determine the substrate specificity of the enzyme
and to carry out the substrate activation(7, 8) .
Despite their structural similarities, remarkable differences in
substrate spectrum are found among the different aromatic ring
dioxygenases(12, 14, 15) . We have focused
on bacteria-degrading chlorinated benzenes, in particular Pseudomonas sp. strain P51. Upon growth on chlorobenzenes,
strain P51 induces enzyme activities which catalyze the conversion of
chlorobenzenes to chlorocatechols(16) . The genes for these
enzymes, the tcbAB genes, were cloned previously from a
catabolic plasmid present in strain P51 and were proposed to encode an
aromatic ring dioxygenase and a dihydrodiol dehydrogenase, similar to
the enzymes of other aromatic pathways(16) . Strain P51 also
contains the genes for a so-called chlorocatechol oxidative pathway,
which in strain P51 consists of an operon of four genes, tcbCDEF(17) , and a regulatory gene tcbR(18) . Interestingly, both gene clusters in strain
P51 are located on a transmissible plasmid pP51. Furthermore, the tcbAB genes itself are part of a transposable element,
Tn5280( To test this idea further,
we wanted to characterize the genes for the chlorobenzene dioxygenase
and for the cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 in detail. We wanted to analyze
further the capability of the enzymes to convert several different
aromatic compounds. This would make it possible to compare the enzymes
both genetically and biochemically with related enzymes from other
aromatic degradation pathways. The data in the paper indicate clearly
that the chlorobenzene dioxygenase and the cis-chlorobenzene
dihydrodiol dehydrogenase resemble most the toluene/benzene-type
enzymes. Furthermore, evidence is presented to show the relics of the
horizontal gene transfer events which may have lead to the imprecise
excision of a genetic element containing the genes for an aromatic ring
dioxygenase and dihydrodiol dehydrogenase from a tod-like
operon.
Figure 1:
Physical map of the
region of the tcbAB genes and of the different cloned DNA
fragments described in this study. The different hatchings in the
physical map indicate the size and location of the ORFs derived from
the DNA sequence analysis. The black ends on both sides of the
physical map indicate the start of the insertion elements, IS1066 (left) and IS1067 (right). The arrows below show the size and location of the DNA sequences
which were determined from the M13mp18-derived subclones. Relevant
restriction sites are indicated on the DNA, but not all vector-located
restriction sites are shown. Restriction sites within brackets point to the NcoI sites which were introduced by PCR to
facilitate cloning. The open arrows in front of the cloned DNA
fragments symbolize the different promoters of the used vector, i.e. T7, T7 promoter of pET8c; lac, lac promoter of pUC18.
The pET8c-derived plasmids did not, unfortunately, express active
Tcb enzymes in E. coli (see below). Therefore, we constructed
a clone containing the complete tcbAaAbAcAd gene sequence in
pUC19. Plasmid pTCB115 was hereto cut with MluI and BamHI, and a 4.7-kb fragment was isolated and ligated with a
3.0-kb MluI-BamHI fragment of pTCB60. After
transformation, this resulted in plasmid pTCB130. We then removed the
complete 4.2-kb XbaI-BamHI insert of plasmid pTCB130
and ligated this fragment into pUC19, cut with XbaI and BamHI. This resulted in plasmid pTCB144. Colonies of E.
coli containing this plasmid turned blue green when growing on LB
plates at 30 °C, which indicates synthesis of active dioxygenase in
the cells(11) .
cis-Chlorobenzene dihydrodiol dehydrogenase activity was
tested in E. coli DH5
Dihydrodiol intermediates were extracted from supernatants of the
whole cell incubations after 2 h with an equal volume of ethyl acetate
and dried with sodium sulfate. Samples were derivatized with BSTFA and
subjected to GC-MS analysis as described elsewhere(22) .
Figure 2:
DNA sequence of the coding strand of the
5,451-bp SphI-MluI fragment containing the tcbAB genes and the predicted amino acid translation of the ORFs
encoding the individual protein subunits. Relevant restriction sites
are indicated, as well as DNA sequences which could function as
ribosome binding sites (in bold). The putative start codon of
the ORF with homology to the sequence of todE is shown
downstream of tcbB. The sequence in italics, starting
at position 5,338, indicates the border repeat sequence of
IS1067. The stop codon, which is introduced by the insertion
of IS1067 in this ORF, is shown underlined at
position 5,353.
Figure 3:
PILEUP clustering of an amino acid
sequence alignment predicted from the gene sequence of a number of
aromatic ring dioxygenases for the terminal oxygenase large subunits
(at a gap creation penalty of 3.0 and a gap extension cost of
0.1)(24) . The right part of the figure shows the
genetic organization of the aromatic ring dioxygenases and surrounding
genes. Horizontal bars indicate the size and location of the
ORFs on the DNA of these organisms. Similar hatchings and shadings represent homologous genes and derived gene products.
Symbols: checkered box, large subunit of the terminal
oxygenase (ISP
It
is interesting that to a large extent the gene organization within the
clusters has been conserved as well, but clearly differs between them.
For example, the benzene/toluene family has the gene order: large
subunit of the terminal oxygenase, small subunit, ferredoxin,
reductase, and, in three cases, dehydrogenase. The group of the
Gram-positive biphenyl dioxygenases, which appeared to be the cluster
closest to that of the benzene/toluene dioxygenases, has an identical
organization of the genes encoding the core dioxygenase, but differs in
genes located downstream. The family of the Gram-negative biphenyl
dioxygenases has a genetic organization comparable to that of the
benzene/toluene family, but in two cases contain an extra ORF between
the genes for the terminal oxygenase and that for the ferredoxin. Also
in these cases, the upstream regions lack homology between each other
or with the corresponding regions of the benzene/toluene family. The
biphenyl dioxygenase of strain KKS102 lacks the extra ORF, but also
lacks the gene for the reductase at this position (Fig. 3). A
stronger difference is found with the family of the naphthalene
dioxygenases. Here the gene order is reductase, ferredoxin, large
subunit of the terminal oxygenase, small subunit, dehydrogenase. In the dox-encoded system, no (clustered) gene for a reductase was
described(29) , but this may similarly be located directly
upstream. The naphthalene reductases (i.e. nahAa and pahAa) are also substantially shorter than those of the others
(329 amino acids versus approximately 410) and biochemically
different (7) . In the case of the benzoate and toluate
dioxygenase, the largest difference in gene order with the others is
found in the presence of a gene for the combined ferredoxin and
reductase function (i.e. benC and xylZ)(8, 9) .
Figure 4:
SDS-PAGE of the cell extracts from E.
coli BL21(DE3) strains containing the different plasmids with tcbAB genes. Lanes: 1, pET8c; 2, pTCB147 (tcbAb, tcbAc, and deletion of tcbAa); 3, pTCB120 (tcbAc, tcbAd, and tcbB); 4, pTCB117 (tcbAc and a deletion of tcbAd); 5, pTCB116 (tcbAc, tcbAd,
and a deletion of tcbB); 6, pTCB115 (tcbAa, tcbAb, tcbAc, and small region of tcbAd); 7, pTCB114 (tcbAa and tcbAb with a
frameshift mutation); 8, pTCB113 (tcbAa). Symbols: Aa, gene product of tcbAa; Aa
Whole cells of E. coli (pTCB144) were incubated with different aromatic substrates in
minimal medium in order to produce the cis-dihydrodiols. E. coli (pTCB144) cells rapidly produced one single metabolite
as detected by HPLC, when incubated with 1,2-dichlorobenzene, toluene,
biphenyl, or naphthalene (Fig. 5). No conversion of benzoate was
detected with these cells. The UV spectra of the intermediates of
toluene, naphthalene, and biphenyl incubation on HPLC were in agreement
with the
Figure 5:
Formation of dihydrodiol intermediates by
washed whole cells of E. coli (pTCB146). Shown are mean values
of peak areas of the intermediates as measured on HPLC from two
independent incubations. Formation of the dihydrodiols excreted in the
supernatant was measured as an increase in absorbance at the wavelength
of the respective absorption maximum, i.e. 262 nm for
naphthalene dihydrodiol, 265 nm for toluene dihydrodiol, 272 nm for
1,2-dichlorobenzene dihydrodiol, and 303 nm for biphenyl
dihydrodiol.
Figure 6:
Electron ionization mass spectra of the
trimethylsilyl derivatives of the products of whole cell incubations
with E. coli (pTCB144) and the following substrates. A, 1,2-dichlorobenzene; B, toluene; C,
biphenyl; D, naphthalene. The structural formula of the
proposed product has been drawn above the respective mass spectrum. The
fragmentation pattern is discussed in the
text.
The dihydrodiols were then incubated with cell extracts
of E. coli (pTCB149), which expresses the dihydrodiol
dehydrogenase, and analyzed by HPLC. In the case of
1,2-dichlorobenzene, we found that the dihydrodiol was converted to one
single product. This product cochromatographed with authentic
3,4-dichlorocatechol, and the UV spectra of the two compounds were
identical. Biphenyl dihydrodiol was enzymatically converted to a
compound with an identical retention time and UV spectrum as
2,3-dihydroxybiphenyl and, similarly, toluene dihydrodiol to a compound
with identity to 3-methylcatechol. In the case of naphthalene
1,2-dihydrodiol, the presumed product 1,2-dihydroxynaphthalene could
not be detected, as it became autooxidized quickly as described
earlier(39, 40) .
Our
studies with the Tcb dioxygenase showed that it is not specific for
catalyzing the conversion of 1,2-dichlorobenzene only, but capable of
converting toluene, naphthalene, and biphenyl. Benzoate was not
converted by the Tcb dioxygenase, which is like other characterized
three-component aromatic ring dioxygenases(8, 12) .
The outcome of the whole cell incubations suggests that naphthalene and
biphenyl are converted even faster than 1,2-dichlorobenzene and
toluene. However, we do not know in what way solubility of these
compounds, different uptake, and excretion rates influence this
outcome. We cannot exclude that other products, such as other
stereoisomers or monohydroxylated rings, were formed in the
incubations, since no attempts were made to determine a mass balance in
the whole cell incubations. Furthermore, we did not determine the
absolute stereoconfiguration of the products. HPLC analysis, however,
suggested the formation of single intermediates. UV and mass spectrum
of these compounds were in agreement with the structures of cis-dihydrodiols, as published
previously(36, 37, 38) . In the subsequent
enzyme incubations, we obtained good evidence that the TcbB dihydrodiol
dehydrogenase converts all of these cis-dihydrodiols to
dihydroxy intermediates (i.e. 3,4-dichlorocatechol,
3-methylcatechol, 2,3-dihydroxybiphenyl, and 1,2-dihydroxynaphthalene).
These results strongly suggest that the formation of dihydrodiols and
dihydroxy compounds by the Tcb dioxygenase and TcbB dihydrodiol
dehydrogenase proceed as expected from the general line for
dioxygenation(7, 8, 12) . It is not so
clear which subunit of the aromatic ring dioxygenases determines the
substrate specificity of the enzyme and why the toluene/benzene
subclass enzymes have such a wide substrate spectrum. The remarkable
potential of the Tod enzyme to catalyze incorporation of oxygen into a
wide range of aromatic substrates has been well studied and
explored(12, 41) . For instance, the Tod dioxygenase
oxidizes biphenyl, the main substrate of the group of bph-encoded enzymes. On the other hand, biphenyl dioxygenase
from P. pseudoalcaligenes KF707 does not oxidize
toluene(14, 42) . This limitation supposedly arose in
the small subunit of Bph dioxygenase, because when hybrid enzymes
between Tod and Bph were constructed, some were found (e.g. BphA1TodC2AB) that gained both the Bph and Tod substrate range.
The Bph-dioxygenases were mostly studied for their capability to
convert (poly-) chlorinated biphenyls. For example, differences in
polychlorinated biphenyls-congener specificity were found between the
Bph dioxygenase from Pseudomonas sp. strain LB400 and P.
pseudoalcaligenes KF707, which in this case were attributed to
changes in the large subunit of the terminal oxygenase(15) . It
will be interesting to study in more detail whether the Tcb dioxygenase
has acquired any new substrate specificities which enable it to convert
higher chlorinated aromatic compounds more efficiently.
A major gene rearrangement has probably taken place in Pseudomonas sp. strain P51. In this microorganism the genes
for the aromatic ring dioxygenase and the dihydrodiol dehydrogenase
were most likely transposed from their original position by the
activity of two insertion elements(19) . The relics of this
process can still be seen on the DNA (Fig. 7). Upstream of the
gene tcbAa there are parts of a gene similar to todF(46) , and downstream of tcbB there is a large
part of a gene for a meta-cleavage enzyme similar to todE(23) . It may have been a necessity for the
organism to inactivate such a meta-cleavage enzyme since these
are known in some microorganisms to interfere with the metabolically
productive ortho-cleavage of chlorinated catechols, which
arise as intermediates in chlorobenzene degradation(47) . We
believe that the strong genetic and biochemical similarity between the tcbAB and the tod system is good evidence to assume
that the tcbAB genes originated in a microorganism which could
use toluene or benzene as the sole carbon and energy source.
Figure 7:
Comparison of the tcbAB genes and
part of the tod gene clusters. Shown is a physical map of the
most important features in this comparison. Blocks indicate
size and location of ORFs on the DNA encoding the different proteins.
Similar shadings or hatchings mean homologous regions
in both gene clusters. Directly upstream of tcbAa there are 35
bp (indicated in black) with homology to the upstream region
of todC1. Some 30 bp further upstream is a region of 201 bp
with homology to todF, although from the start of this gene.
Percentages of identity in nucleotide sequence between the different
gene regions are given in the figure. Downstream of tcbB is
the part of the gene with homology to todE. The sizes and
locations of the ORFs for tcbAb, tcbAc, and tcbAd,
respectively, todC2, todB, and todA are not shown in
detail here.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
U15298[GenBank].
Volume 271,
Number 8,
Issue of February 23, 1996 pp. 4009-4016
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
)(19) . We therefore strongly
believe that the chlorobenzene pathway is an evolutionary novelty in
bacteria, formed by a novel combination of two existing gene clusters,
perhaps through horizontal gene transfer.
Bacterial Strains, Plasmids, and Growth
Conditions
Pseudomonas sp. strain P51 has been
described previously(16) . Escherichia coli strains
DH5
and TG1 were used routinely for plasmid cloning and
single-stranded M13 phage preparation, respectively(20) . E. coli BL21(DE3) was used for T7 RNA polymerase-directed
expression of genes and gene fragments cloned in plasmid
pET8c(21) . We used the plasmids pUC18 and pUC19 (obtained from
Boehringer Mannheim, Mannheim, Federal Republic of Germany) as cloning
vectors for strain P51-derived DNA fragments. In general, E. coli strains were grown on LB medium at 37 °C (20) ,
supplemented with the appropriate antibiotics. For expression of active
chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol
dehydrogenase, however, the E. coli strains were cultivated at
25 °C.DNA Techniques and Sequence Analysis
All DNA
techniques, such as plasmid DNA isolation, transformations, or
DNA-enzyme digestions, were carried out according to established
procedures described elsewhere(20) . DNA sequence analysis was
performed on both strands of the DNA by sequencing overlapping
fragments cloned in M13mp18, as described elsewhere(17) . The
source of the DNA fragment containing the chlorobenzene dioxygenase and
dihydrodiol dehydrogenase genes of strain P51 was plasmid
pTCB60(16) . Restriction enzymes and other DNA-modifying
enzymes were purchased from Life Technologies Europe (Paisley, UK),
Appligene (Illkirch, France), or Boehringer Mannheim (Mannheim, FRG).
Reagents for the polymerase chain reaction were obtained from Life
Technologies Inc.Construction of tcb Expression Clones
To obtain
overexpression of the individual components of the dioxygenase system
and the dehydrogenase, we constructed a number of translational fusions
with the start codon present on pET8c (Fig. 1)(21) .
Hereto, artifical NcoI sites were created on the DNA to be
cloned by applying PCR amplification in the presence of a mutagenic
primer. For clones starting with the tcbAa gene, we amplified
a 200-bp DNA fragment ranging from the start of tcbAa until
the first SphI site downstream. The PCR fragment was then
cleaved with NcoI and SphI and ligated with a 2-kb SphI-ScaI fragment of plasmid pTCB71 (16) and, with pET8c, cut with NcoI and EcoRV. After transformation, this resulted in plasmid pTCB113.
The sequence of the PCR-amplified part of this plasmid was determined
and found to be identical with that of the wild-type tcbAa gene. From plasmid pTCB113 we then constructed pTCB115 by
exchanging the 1.2-kb MluI-ScaI fragment from pTCB113
with the 2.0-kb MluI-PstI fragment, which contains tcbAb, tcbAc, and part of tcbAd. In plasmid
pTCB114 the frame of the tcbAb gene was interrupted by cutting
the DNA with AatII, filling the ends by using Klenow enzyme,
and religating. Plasmid pTCB147 was constructed from pTCB115 by cutting
with SacII, removing the 1.0-kb fragment, creating blunt ends
with T4 DNA polymerase treatment, and religating. Clones starting with
the tcbAc gene were made by introducing a NcoI site
at the start codon of tcbAc similarly as described above. A
small region of the DNA was amplified by PCR between the start codon of tcbAc and the first downstream SalI site, then
digested with NcoI and SalI, and ligated with a
1.3-kb SalI-EcoRI fragment containing the region of tcbAc and tcbAd and with pET8c, cut with NcoI and EcoRI. This resulted in plasmid pTCB117. A
DNA fragment containing the complete tcbAd and tcbB sequence was then introduced in plasmid pTCB117 to form pTCB120. A
deletion derivative was created which lacked the complete tcbB sequence by cutting this plasmid with BamHI and SacI, removing protruding ends, and religating (pTCB116).
Finally, a clone containing only tcbB was made by cloning a
1.2-kb EcoRI-MroI fragment of pTCB119 in pUC19 cut
with EcoRI and SmaI. This plasmid was named pTCB149.
Overproduction of Individual Components of the
Dioxygenase and Dehydrogenase
To test if the observed ORFs could
be translated to proteins of the predicted size, the pET8c-derived
plasmids containing the various parts of the tcb genes were
tested for expression in E. coli BL21(DE3). These cultures
were grown on LB medium at 37 °C to an A of
0.5. We then induced T7-mediated gene expression by adding
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM to the cell culture. Cultures were then
allowed to grow for another 2 h, after which the cells were harvested
from a 1-ml sample. The obtained cell pellet was resuspended in 50
µl of protein sample buffer according to Laemmli(50) , and
5-10 µl were analyzed on 12.5% SDS-PAGE.Analysis of Chlorobenzene Dioxygenase and Chlorobenzene
Dihydrodiol Dehydrogenase Activity
Chlorobenzene dioxygenase
activity was tested by analyzing the formation of dihydrodiol
intermediates from aromatic substrates incubated with washed cell
suspensions of E. coli DH5 (pTCB144). The cultures were
inoculated from a single colony in 200 ml of LB medium with 50
µg/ml ampicillin and grown at 25 °C for about 36 h. The optical
density of the cultures had then reached an A of
3.5. The cell culture was then centrifuged at 4000 rpm for 10 min at 20
°C, and cells were resuspended in 50 ml of M9 mineral salts
medium(20) . This was repeated once more, and, after a final
centrifugation step, the cells were resuspended in 10 ml of M9 medium
and stored briefly on ice until use. A series of glass-stoppered tubes
with a volume of 15 ml were filled with the following reagents: 4.5 ml
of M9 containing 1 mM glucose, 50 µl of a methanol
solution containing the aromatic substrates, and 0.5 ml of the cell
suspension. The final A
of the cells in the
assay was between 0.7 and 1.0. The tubes were incubated on a rotary
shaking platform at a temperature of 30 °C. We tested the following
aromatic substrates at a final concentration in the assay of 0.5
mM: 1,2-dichlorobenzene, toluene, biphenyl, naphthalene (all
previously dissolved in methanol), and benzoate. For each time point of
the assay, two tubes were incubated. Samples of 1.0 ml were then taken
from the tubes and centrifuged for 1 min at 13,000 rpm to remove the
cells. The supernatant was transferred to a fresh tube and analyzed for
the presence of dihydrodiol intermediates by HPLC (see below).
(pTCB149). Cultures were grown on
50 ml of LB medium at 37 °C to an A of 1.0,
after which the cells were harvested and a cell extract was prepared as
described previously(16) . The reaction mixture for dihydrodiol
dehydrogenase activity contained 0.65 ml of 20 mM sodium
phosphate buffer, pH 7.5, 25 µl of 20 mM NAD
solution, 50 µl of the cell extract, and 50 µl of
dihydrodiol substrate. As substrates we used the supernatants of the
whole cell incubations after 2 h (see above). The assay mixture was
incubated at 37 °C, and the change in A
was
measured on a spectrophotometer. When no more changes in A
were observed, the assay mixture was analyzed
on HPLC to check for the disappearance of the dihydrodiol and the
presence of the dihydroxy compound.
HPLC and GC-MS Analysis
Analysis of dihydrodiols
and dihydroxy compounds was performed on a Waters 625 LC HPLC system
equipped with a photodiode array detector. Separation was carried out
on a C18 reversed phase column (Nova-Pak 300 mm, 6 nm, 4 µm). Two
running solutions were used which contained: A, 10 mM H
PO
in H
O at a pH of 3.0, and
B, 90% methanol and 10% of solution A. Elution from the column was
performed by running a gradient as follows: 0-2 min, 40% of
buffer B and 60% of buffer A; 2-30 min, linear increase to 70% of
buffer B and decrease of buffer A to 30%; 30-40 min, 70% of
buffer B and 30% of buffer A. Flow-rate through the system was 0.5
ml/min at a pressure of 3500 p.s.i. Generally, an amount of 200 µl
of the samples was injected. Under these conditions we observed the
following retention times:
3,4-dichloro-1,2-dihydroxycyclohexa-3,5-diene (3,4-dichlorobenzene
dihydrodiol), 10.8 min; 3,4-dichlorocatechol, 29.5 min;
1,2-dihydroxy-3-methylcyclohexa-3,5-diene (toluene dihydrodiol), 5.6
min; 3-methylcatechol, 14.8 min; 1,2-dihydroxy-1,2-dihydronaphthalene
(naphthalene dihydrodiol), 13.4 min;
1,2-dihydroxy-3-phenylcyclohexa-3,5-diene (biphenyldihydrodiol), 21.5
min; 2,3-dihydroxybiphenyl, 35.5 min. Authentic standard compounds
which were available to us and could be tested were
2,3-dihydroxybiphenyl, 3,4-dichlorocatechol, and 3-methylcatechol.
Sequence Determination of the tcbAB Genes
We
determined the nucleotide sequence of the region containing the tcbAB genes of Pseudomonas sp. strain P51 on both
strands of the DNA. The tcbAB genes are located on a stretch
of 5,402 base pairs which lay between IS1066 and IS1067(19) ( Fig. 1and Fig. 2). The region showed
the presence of five large unidirectional ORFs, encoding the different
subunits of the chlorobenzene dioxygenase and the dihydrodiol
dehydrogenase. Sequence homologies with other known dioxygenases
allowed the assignment of putative protein functions to each of the
ORFs (Table 1). We propose to designate the genes as follows: tcbAa, coding for the large subunit of the terminal oxygenase; tcbAb, encoding the small terminal oxygenase subunit; tcbAc, the ferredoxin; tcbAd, the NADH reductase; and tcbB, the dihydrodiol dehydrogenase. Except for a small 109-bp
gap between tcbAa and tcbAb and 8 bp between tcbAb and tcbAc, the ORFs were contiguous on the DNA.
Downstream of tcbB, another ORF was found, which showed
homology to catechol 2,3-dioxygenases, such as todE(23) . This ORF, however, appeared to be
interrupted by IS1067, causing a premature ending (Fig. 2).
Homologies with Other Bacterial Aromatic Ring
Dioxygenases and Dihydrodiol Dehydrogenases
The amino acid
sequences predicted for the TcbAa, -Ab, -Ac, -Ad, and TcbB proteins
were compared with those from other bacterial aromatic ring
dioxygenases by using the GCG programs FASTA, DISTANCES, and
PILEUP(24, 25) . The alignments and distance
calculations showed for almost every individual component of the
dioxygenases and for the dihydrodiol dehydrogenase a clustering in four
different families ( Fig. 3and Table 2). One family is
formed by the dioxygenases which are composed of two components (and
three protein subunits), i.e. benzoate dioxygenase of Acinetobacter calcoaceticus(9) and toluate
dioxygenase of Pseudomonas putida(10) . A second
family contains the three-component dioxygenases of naphthalene
metabolism, such as those encoded by the nah(26) , ndo(27) , pah(28) , or dox(29) genes. The third family is composed of the
dioxygenases for biphenyl and chlorobiphenyl conversion in
Gram-negative bacteria(30, 31, 32) , and a
fourth one of the benzene(13, 33) ,
toluene(23) , and chlorobenzene dioxygenases. Two recently
published sequences for a biphenyl dioxygenase from two Gram-positive
microorganisms aligned more closely with the toluene/benzene family
than with the biphenyl family itself (34, 35) (Fig. 3). The only exception in the
alignments was the clustering of the reductase components. In this
case, the positions of the reductases from P. paucimobilis KKS102, Rhodococcus sp. RHA1, and R. globerulus P6 appeared to be intermediate (Table 2). In general, the
reductases seem to have diverged substantially more than the other
components of the dioxygenases and the dihydrodiol dehydrogenase.
); waffled box, small subunit of
the terminal oxygenase (ISP
); dotted box,
ferredoxin; white on black lined box, reductase; diagonally lined box, dihydrodiol dehydrogenase; shaded
box, meta-cleavage enzyme. The figure does not show the
location of all genes within a particular gene cluster. ben genes, Acinetobacter calcoaceticus(9) ; xyl, P. putida mt2(3, 10) ; dox, (29) ; ndo, P. putida NCIB9816(26, 27) ; nah, P. putida G7(26) ; pah,(28) ; bph, P.
pseudoalcaligenes KF707(30, 48) ; PcBph, bph genes of Pseudomonas sp. strain
LB400(32, 49) ; Kks102Bph, bph genes
of P. paucimobilis strain KKS102(31) ; bed, P. putida ML2(33) ; tod, P. putida F1(23, 46) ; tcb, Pseudomonas sp. strain P51; bnz, P. putida 136-R3(13) ; RgBph, bph genes of Rhodococcus globerulus P6(35) ; RsBph, bph genes of Rhodococcus sp.
RHA1(34) .
Expression of the tcb-encoded Gene Products
We
cloned all open reading frames from the tcbAB cluster in the
expression vector pET8c under transcriptional control of the T7
promoter in E. coli BL21(DE3), to test if the gene products
would have the size as predicted from the amino acid sequence. Upon
induction in E. coli, we could detect all predicted protein
bands and deletion derivatives on SDS-PAGE (Fig. 4).
Interestingly, in some cases, read-through from one ORF into the other
occurred only sparsely. For example, the tcbAb gene could not
be visibly expressed in clones containing tcbAa upstream of tcbAb. Only by using plasmids in which part of tcbAa was deleted, such as pTCB147, we found detectable expression of tcbAb. On the other hand, clones starting with tcbAc would also express downstream ORFs when present, such as in
plasmid pTCB116 and pTCB120. Most protein bands observed on SDS-PAGE
which were attributed to expression from a tcb gene were of
the size expected from computer predictions (Table 1). The
exception was TcbB, which migrated at a smaller apparent molecular mass
than predicted (22 kDa instead of 33.1 kDa).
, product of the interrupted tcbAa gene; Ab, product of tcbAb; Ac, product of tcbAc; Ad, product of tcbAd; Ad, product of the interrupted tcbAd; B, product of tcbB; B, product of the
interrupted tcbB gene. Migration of the molecular mass
standards is indicated in kilodaltons on the right
side.
Chlorobenzene Dioxygenase and Dihydrodiol Dehydrogenase
Activity in E. coli
The functionality of the tcbAB gene
products was then tested by measurement of their enzymatic activity in E. coli. We cloned the complete DNA fragment with the tcbAaAbAcAd genes starting at the ATG codon of the tcbAa gene in pET8c. To our surprise, we could not detect any measurable
activity of the chlorobenzene dioxygenase with this plasmid (not
shown). We think that this may be caused by the unbalanced expression
of the different ORFs when expressed from the T7 promoter (see above)
and by the formation of inclusion bodies. The genes were then removed
from pET8c and cloned in pUC19 under control of the lac promoter (pTCB144). E. coli (pTCB144) showed the typical
formation of blue-green colonies when grown on LB agar, due to the
formation of indigo. This color became more pronounced when the
colonies were incubated at 25 °C.
values published previously for the
corresponding dihydrodiols(36, 37, 38) . For
the product of 1,2-dichlorobenzene incubation, we observed a UV
spectrum similar to that of toluene, although with a at 272 nm. Further information on the identity of the four
intermediates was obtained by GC-MS analysis of the BSTFA-derivatized
form (Fig. 6). All four mass spectra gave a similar
fragmentation pattern with the molecular ions showing at m/z 324, 270, 332, and 306 for the products of dichlorobenzene (Fig. 6A), toluene (Fig. 6B), biphenyl (Fig. 6C), and naphthalene (Fig. 6D),
respectively. The usually dominant (M - 15) ion
(loss of one of the methyl groups of the trimethylsilyl moiety) is
absent from all the mass spectra, but loss of OSi(CH
) (molecular mass of 89) is apparent in all of them. The ions at m/z 191
([(CH)SiOCHOSi(CH)]),
147
([(CH
)SiOSi(CH)]),
and 73 ([(CH
)Si])
dominate the four spectra. In the case of the product of
dichlorobenzene, the ion at m/z 289 is formed by the loss of
chlorine (mass of 35) from the molecular ion. Following the line of
evidence for the formation of cis-dihydrodiols from aromatic
compounds by toluene and naphthalene
dioxygenase(36, 37, 38) , for example, all
our results are in agreement with the proposed cis-dihydrodiol
structures which would be formed during conversion of toluene,
1,2-dichlorobenzene, biphenyl, and naphthalene by the chlorobenzene
dioxygenase.
Chlorobenzene Dioxygenase Belongs to the Toluene and
Benzene Dioxygenase Subclass
Pseudomonas sp. strain P51
has the ability to use chlorinated benzenes as sole carbon and energy
source. The enzyme catalyzing the initial dioxygenation of the aromatic
ring structure was presumed to be a three-component aromatic ring
dioxygenase, like those found in other aerobic bacteria. Here we have
shown that the genes for the chlorobenzene dioxygenase are contiguous
on the DNA and indeed code for four protein subunits, two of which make
up the terminal oxygenase, one the ferredoxin, and the last one the
NADH reductase. Following the genes for the dioxygenase is a gene
coding for a dihydrodiol dehydrogenase. All genes were shown to be
functional by using expression studies and enzyme activity assays. Both
biochemical and genetic evidence indicate that the chlorobenzene
dioxygenase belongs to a subclass of aromatic ring dioxygenase enzymes
to which the toluene and benzene dioxygenases also belong.Gene Rearrangements in the Evolutionary Divergence of
Aromatic Ring Dioxygenases
The large pool of genetic data on
aromatic ring dioxygenase systems from different aerobic bacteria makes
it possible to speculate about the different events which have taken
place in the course of the evolutionary development of these
microorganisms(43) . The accumulation of small events (e.g. mutations) has likely led to the divergence of the different
individual genes and gene products, as shown in Fig. 3. However,
more striking larger genetic changes have also occurred. Rearrangements
on the DNA have caused differences in gene order of the aromatic ring
dioxygenase. This becomes obvious when we compare the gene order of the
toluene/benzene family and the biphenyl family on one side, and that of
the naphthalene family on the other (Fig. 3). In the naphthalene
family, the genes for the reductase and ferredoxin have inverted their
position with respect to the genes encoding the terminal oxygenase. The
gene for the reductase may also be at a different position, as in the bph gene cluster of P. paucimobilis strain
KKS102(31, 44) . Similarly, rearrangements have caused
differences in the organization of genes located downstream of the
aromatic ring dioxygenase, like the ones encoding the dihydrodiol
dehydrogenase or the meta-cleavage enzymes (Fig. 3).
These DNA rearrangements must have had their effects on gene expression
and on enzyme synthesis, perhaps due to improper signals on the DNA or
changed stability and structure of the mRNA. For instance, how is it
achieved that the right molar proportions of all components of the
dioxygenase are synthesized? The benzene dioxygenase of P. putida ML2, which is transcribed from one single gene cluster with
operonic organization, apparently has intracellular molar proportions
of 1:0.45:0.8 of ISP- subunit/ferredoxin/reductase(45) .
Regulation of the right molar amounts may become less obvious when the
reductase gene is not directly transcriptionally coupled. This may
reflect the idea that the aromatic ring dioxygenase is a kinetic enzyme
complex with a rather loose association between oxygenase component,
ferredoxin, and reductase (45) . For the tcbAB genes
it was interesting to notice that we could not see expression of tcbAb in E. coli under control of the T7 RNA
polymerase from plasmid constructs on which tcbAa was still
present, although expression was clearly forced upon the system in this
case.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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