JOURNAL OF BACTERIOLOGY, Aug. 2009, p. 4905–4915
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 15
Characterization of a Gene Cluster Involved in 4-Chlorocatechol
Degradation by Pseudomonas reinekei MT1?
Beatriz Ca ´mara,1† Patricia Nikodem,1‡ Piotr Bielecki,2Roberto Bobadilla,3
Howard Junca,1§ and Dietmar H. Pieper1*
Department of Microbial Pathogenesis1and Division of Molecular Biotechnology,2HZI Helmholtz Centre for
Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany, and Departamento de Prevencio ´n de
Riesgos y Medio Ambiente Universidad Tecnolo ´gica Metropolitana, Dieciocho No. 390, Santiago, Chile3
Received 10 March 2009/Accepted 17 May 2009
Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway
with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a
gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report
that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12OccaA, a novel
(chloro)muconate cycloisomerase, MCIccaB, which showed features not yet reported, was induced. This cyclo-
isomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates
and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that
it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The
corresponding genes, ccaA (C12OccaA) and ccaB (MCIccaB), were located in a 5.1-kb genomic region clustered
with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative
regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes
sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that
C12OccaAand MCIccaBare only distantly related to previously described catechol 1,2-dioxygenases and
muconate cycloisomerases. Kinetic analysis indicated that MCIccaBand the previously identified C12OsalD,
rather than C12OccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a
completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding
enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and
The aerobic degradation of chloroaromatic compounds usu-
ally proceeds via chlorocatechols as central intermediates (20,
47), which in most of the cases reported thus far, are further
degraded by enzymes of the chlorocatechol pathway (44). This
pathway involves ortho-cleavage by a chlorocatechol 1,2-dioxy-
genase with high activity for chlorocatechols (12), a chloromu-
conate cycloisomerase with high activity for chloromuconates
(54), a dienelactone hydrolase active with both cis- and trans-
dienelactone (4-carboxymethylenebut-2-en-4-olide) (54), and a
maleylacetate reductase (MAR) (28).
However, it has become evident in recent years that micro-
organisms have evolved various alternative strategies to min-
eralize chlorocatechols. Pseudomonas putida GJ31 was found
to degrade chlorobenzene rapidly via 3-chlorocatechol using a
catechol meta-cleavage pathway (33). Two alternative path-
ways for 3- and 4-chlorocatechol degradation that involve re-
actions known from the chlorocatechol, as well as the 3-oxo-
adipate, pathway have recently been observed in Rhodococcus
opacus 1CP (35) and Pseudomonas reinekei MT1 (39). In R.
opacus 1CP, 3-chloro- and 2,4-dichloro-cis,cis-muconate (the
ring cleavage products of 4-chlorocatechol and 3,5-dichloro-
catechol, respectively) are converted to the respective cis-
dienelactones (35, 58), similar to the reaction described for
proteobacterial chloromuconate cycloisomerases (54). How-
ever, proteobacterial chloromuconate cycloisomerase can de-
halogenate 2-chloromuconate (the ring cleavage product of
3-chlorocatechol) and transform this compound via 5-chloro-
muconolactone into trans-dienelactone (54, 65), whereas none
of the described chloromuconate cycloisomerases of R. opacus
1CP can catalyze such a dehalogenation, and 5-chloromucono-
lactone is the product of the cycloisomerization reaction (35,
58). Dehalogenation is achieved by an enzyme with high se-
quence similarity to muconolactone isomerases (35), which in
proteobacteria have been shown to be capable of dehalogenat-
ing 5-chloromuconolactone to cis-dienelactone (46).
In P. reinekei MT1, a trans-dienelactone hydrolase (trans-
DLH) was identified as the key enzyme involved in the degra-
dation of 4- and 5-chlorosalicylate via 4-chlorocatechol as an
intermediate (39). In contrast to all previously described
dienelactone hydrolases involved in chlorocatechol degrada-
tion, which belong to the ?/? hydrolase fold enzymes with a
catalytic triad consisting of Cys, His, and Asp (10), trans-DLH
was shown to be a zinc-dependent hydrolase (8). The function
* Corresponding author. Mailing address: Department of Microbial
Pathogenesis, HZI Helmholtz Centre for Infection Research, Inhof-
fenstraße 7, D-38124 Braunschweig, Germany. Phone: (49) 531 6181
4200. Fax: (49) 531 6181 4499. E-mail: email@example.com.
† Present address: Department of Microbiology and Centre for Mo-
lecular Microbiology and Infection, Division of Investigative Sciences,
Flowers Building, Imperial College London, London SW7 2AZ,
‡ Present address: Novo Nordisk A/S, Hallas Alle ´e, 4400 Kalund-
§ Present address: Centro Colombiano de Geno ´mica y Bioinforma ´tica
de Ambientes Extremos (GeBiX), Grupo de Gene ´tica Molecular, Cor-
poracio ´n CorpoGen, Carrera 5 No. 66A-35, Bogota ´, Colombia.
?Published ahead of print on 22 May 2009.
of this enzyme in the 4-chlorocatechol metabolic pathway was
to interact with the muconate cycloisomerase (MCI)-mediated
transformation of 3-chloromuconate into protoanemonin. By
acting on the reaction intermediate 4-chloromuconolactone,
trans-DLH prevents the formation of protoanemonin by cata-
lyzing its hydrolysis to maleylacetate (39). Maleylacetate, in
turn, is reduced by MAR to 3-oxoadipate.
A more detailed genetic and biochemical analysis of the
degradation of differently substituted salicylates (7) had shown
the presence of two catabolic gene clusters in MT1. An arche-
type catRBCA gene cluster was shown to be involved in salic-
ylate degradation. The second gene cluster (sal) had a novel
gene arrangement, with salA, encoding a salicylate 1-hydroxy-
lase, clustered with the salCD genes, encoding MCI and cate-
chol 1,2-dioxygenase (C12O), respectively. As these genes
were expressed during growth on differently substituted salicy-
lates, it was proposed that the function of the sal gene cluster
is to channel both chlorosubstituted and methylsubstituted sa-
licylates into a catechol ortho-cleavage pathway, followed by
dismantling of the formed substituted muconolactones through
specific pathways. However, previous analyses had indicated
the presence of an additional and thus third (chloro)muconate
cycloisomerase in MT1 during growth on chlorosalicylate,
which is distinct from both previously described MCIs encoded
by the cat cluster (MCIcatB) and the sal cluster (MCIsalC), as it
amounts of cis-dienelactone and protoanemonin (39). In the
present report, this cycloisomerase is biochemically and genet-
ically described and shown to be located in a third gene cluster
involved in the degradation of 5-chlorosalicylate by strain
MT1. This cluster comprises genes encoding a third C12O,
trans-DLH (8), and a MAR. Evidently, P. reinekei MT1 is the
first microorganism in which such a complex net of genes
involved in chlorocatechol degradation has been described.
MATERIALS AND METHODS
Bacterial strain and culture conditions. P. reinekei MT1 was grown and cell
extracts were prepared as previously described (39).
Enzyme assays. C12O, MCI, trans-DLH, and MAR activities were determined
spectrophotometrically as previously described (27, 39, 54). The activity of
MCIccaBwith 3-chloromuconate was determined by high-performance liquid
chromatography (HPLC) (39) following substrate depletion and product forma-
tion. To more sensitively follow the activity of MCIccaBwith muconate and
2-chloromuconate, the transformation of these substrates (100 ?M) was also
followed by HPLC using up to 10 U/ml (measured with 100 ?M 3-chloromu-
conate) of purified MCIccaB. Specific activities are expressed as ?mol of substrate
converted or product formed per minute per gram of protein at 25°C. Protein
concentrations were determined by the Bradford procedure using the Bio-Rad
protein assay with bovine serum albumin as a protein standard (5).
Analysis of kinetic data. The Vmax, kcat, and apparent Kmvalues of C12OccaA
with catechol, 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol were
determined using 1 to 100 ?M of substrate in air-saturated buffer, and the kinetic
data were calculated from the initial velocities using the Michaelis-Menten equa-
tion by nonlinear regression (KaleidaGraph; Synergy Software). As very low Km
values were indicated by this method, kinetic data were finally determined from
progress curves obtained from reactions with initial substrate concentrations of
10 ?M, as previously described (7). Vmax, kcat, and apparent Kmvalues of
MCIccaBwith 2-methylmuconate, and 3-methylmuconate were determined using
2 to 100 ?M of substrate. Transformation of 3-chloromuconate was determined
by HPLC analysis at substrate concentrations of 50 ?M to 500 ?M. Samples were
taken during the reaction time, and the formation of protoanemonin and cis-
dienelactone was directly quantified by HPLC analysis. At least two independent
experiments were performed for each value. Kmand Vmaxvalues were calculated
by nonlinear regression to the Michaelis-Menten equation, using KaleidaGraph
(Synergy Software). Turnover numbers (kcatvalues) were calculated assuming
subunit molecular masses of 29,424 (C12OccaA) and 39,764 (MCIccaB) Da,
Enzyme purification. C12OccaAand MCIccaBwere purified using a Fast Pro-
tein Liquid Chromatography system (Amersham Biosciences). Cells were har-
vested during late exponential growth with 5-chlorosalicylate or 4-methylsalicy-
late. Cell disruption and all protein elutions were performed in 50 mM Tris-HCl,
pH 7.5, 2 mM MnCl2.
For analyzing the presence and abundances of different C12Os and MCIs
under different growth conditions, either cell extracts (usually containing 35 mg
of protein per ml) were applied directly to a MonoQ HR5/5 (Amersham Phar-
macia Biotech) and proteins were eluted by a linear gradient of 0 to 0.5 M NaCl
over 25 ml with a flow of 0.5 ml/min, or the cell extract was mixed with 4 M
(NH4)2SO4to give a final concentration of 1 M (NH4)2SO4and applied to a
Source 15PHE PE 4.6/100 (hydrophobic interaction) column (Amersham Phar-
macia Biotech). Proteins were eluted from the Source column by a linear gra-
dient of (NH4)2SO4(1 M to 0 M) over 25 ml with a flow of 0.5 ml/min. Fraction
volumes were 0.5 ml. Hydrophobic interaction chromatography (HIC) separated
C12OccaA(0.52 ? 0.02 M), C12OsalD(0.45 ? 0.04 M), C12OcatA(0.16 ? 0.04
M), MCIccaB(0.25 ? 0.04 M), MCIsalC(0.06 ? 0.06 M), and MCIcatB(0.12 ?
0.06 M), thus excluding interference between their activities. During anion-
exchange chromatography, C12OccaAeluted at 0.23 ? 0.01 M NaCl, whereas
MCIccaBeluted at 0.37 ? 0.02 M NaCl. Under these conditions, C12OcatAand
C12OsalDhad been shown to coelute at 0.28 ? 0.02 M NaCl, whereas MCIcatB
and MCIsalCcoeluted at 0.24 ? 0.02 M NaCl (7).
For purification of C12OccaA, 35 mg of protein from 5-chlorosalicylate-grown
cells was applied to the MonoQ HR 5/5 (Amersham Pharmacia Biotech), and
proteins were eluted as described above. Fractions containing C12OccaAactivity
were combined, supplemented with 4 M (NH4)2SO4to give a final concentration
of 1 M (NH4)2SO4, and loaded on a Source 15PHE PE 4.6/100 (hydrophobic
interaction) column (Amersham Pharmacia Biotech) as described above.
For purification of MCIccaB, up to 400 mg of protein from 5-chlorosalicylate-
grown cells was applied to a MonoQ HR 10/10 (Amersham Pharmacia Biotech).
A stepwise gradient of 0 to 60 mM NaCl over 40 ml, 60 to 380 mM NaCl over 120
ml, and 380 to 2,000 mM NaCl over 40 ml was applied. The flow rate was 0.3
ml/min. The eluate was collected in fractions of 5 ml. All fractions eluting at
NaCl concentrations of 90 to 330 mM were pooled and concentrated to a final
volume of 4.25 ml using ultrafiltration by Centriprep YM-50 (Millipore) accord-
ing to the protocol of the manufacturer. The protein solution was supplemented
with 4 M (NH4)2SO4to give a final concentration of 0.8 M (NH4)2SO4and
centrifuged directly before application of the soluble proteins to the Source
column. Aliquots comprising 40 mg of protein were separated as described
above. Fractions containing MCIccaBwere combined and concentrated by a
Centricon YM-50 (Millipore). Further purification was achieved by gel filtration
using a Superose 12 HR10/10 column (Amersham Pharmacia Biotech). Proteins
were eluted with 50 mM Tris-HCl, 2 mM MnCl2, pH 7.5, over 15 ml (flow rate,
0.2 ml/min; fraction volume, 0.5 ml). The fractions containing high MCIccaB
activity (eluting at 10.5 to 11.5 ml) were applied to a MonoQ HR5/5 (anionic-
exchange) column (Amersham Pharmacia Biotech), and the proteins were eluted
by a linear gradient of 0 to 0.4 M NaCl over 25 ml with a flow of 0.2 ml/min.
Homogeneity was verified by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE). trans-DLH was purified as previously described (8).
Transformation of 3-chloromuconate by enzyme mixtures. Product formation
from 3-chloromuconate by purified MCIccaBand in the presence of purified
trans-DLH was analyzed by HPLC in assays performed at room temperature in
150 ?l Tris-HCl (50 mM), 2 mM MnCl2, pH 7.5, with 120 ?M 3-chloromuconate
as a substrate. MCIccaBwas added to give an activity of 53 mU/ml (determined
by the transformation of 100 ?M 3-chloro-cis,cis-muconate), corresponding to
8.8 nM MCIccaB, whereas trans-DLH was applied in amounts ranging from 1.32
to 1,320 mU/ml (determined by the transformation of 50 ?M trans-dienelac-
tone), corresponding to 0.88 to 88 nM trans-DLH.
Determination of molecular mass. The molecular mass of MCIccaBwas de-
termined by gel filtration using a Superose 12 column as described above. The
column was calibrated for molecular mass determinations using ovalbumin (43
kDa), aldolase (158 kDa), catalase (232 kDa), and ferritin (440 kDa) from
Electrophoretic methods. SDS-PAGE was performed on a Bio-Rad Minipro-
tein II as previously described (32), with acrylamide concentrations of 5 and 10%
(wt/vol) used for the concentrating and separating gels, respectively. The pro-
teins were stained with Coomassie brilliant blue (Serva). A PageRuler Protein
Ladder (Fermentas) was used as a marker.
Amino acid sequencing. N-terminal amino acid sequences were determined as
described previously (26).
4906CA ´MARA ET AL. J. BACTERIOL.
Identification of the gene encoding MCIccaBof strain MT1. Part of the gene
encoding MCIccaBwas amplified by PCR using the degenerate primers NT1B
(WSNCARGGNTTYGTNATCGG) and NTREV2A (AANWSCATNCKDAT
NGGCTG), which were designed based on the determined N-terminal protein
sequence (underlined) SQGFVIGRVLAQRLDIPFSQPIRMSFGTLD. Touch-
down PCR consisted of an initial denaturation (94°C for 4 min), followed by 10
cycles of denaturation (94°C for 45 s), annealing (60°C for 30 s ? 1°C per cycle),
and elongation (72°C for 30 s), followed by 25 cycles with an annealing temper-
ature of 50°C for 45 s and a final elongation step (72°C for 7 min). A 72-bp
fragment was obtained, cloned into the pGEM-T Easy vector (Promega), and
transformed into Escherichia coli XL10-Gold (Stratagene), and inserts of the
clones generated were then sequenced. The deduced amino acid sequence
matched that of the N-terminal amino acid sequence.
An extended part of the gene encoding MCIccaBwas amplified by PCR using
the primers MCIB1 (GCAACGGCTGGATATACCTT) and MCIBR2 (GTRT
CGCCRCTSGCSARCGTCC), which were designed based on the DNA se-
quence generated above and a protein sequence, WTLASGDT, identified by
protein sequence alignment to be conserved in proteobacterial muconate and
chloromuconate cycloisomerases. The touchdown PCR conditions included 10
cycles as described above, followed by 25 cycles at an annealing temperature of
55°C. An approximately 400-bp fragment was obtained, cloned, and sequenced as
mentioned above. The DNA sequence matched the sequence deduced from the
N-terminal sequence, clearly confirming that the cloned PCR product corre-
sponded to part of the gene encoding MCIccaB.
DNA isolation, fosmid library construction, and identification of the cca gene
cluster. Preparation of the fosmid library in pCC1FOS, which comprised a total
of 282 individual clones, was previously described (7). The fosmid library was
screened by PCR using primers specific for ccaB (MCIB1 [GCAACGGCTGG
ATATACCTT] and inMCIB [AGCAGAAACACCCAACTGCT], with an an-
nealing temperature of 59°C). Fosmid clones harboring the expected 340-bp
ccaB gene fragment were subsequently checked by PCR for the presence of the
ccaC gene, encoding trans-DLH (TransFOR [AATCCCTGCCGACATACA
AG] and TransREV [CGTCAGCATGAAGGTGTAGC]). From the three fos-
mids carrying both the ccaB and the ccaC gene fragments, one fosmid was chosen
and purified with the FosmidMAX DNA purification kit (Epicentre), and the
complete cca gene cluster was obtained by direct sequencing (Seqlab, Go ¨ttingen,
Germany) from the purified fosmid with a sixfold coverage of the insert.
DNA sequencing and sequence analysis. PCR products were purified with the
QIAquick PCR Purification Kit (Qiagen) and sequenced using the ABI Prism
BigDye Terminator v1.1 Ready Reaction Cycle Sequencing Kit (Applied Bio-
systems) and a DNA capillary sequencer, the 3130xl Genetic Analyzer (Applied
Biosystems). Raw sequence data from both strands were assembled with Se-
quencher software version 4.0.5 (Gene Codes Corporation). DNA and protein
similarity searches were performed using the BLASTN and BLASTP programs
from the NCBI website. Translated protein sequences were aligned with
CLUSTALX 1.83 using default values (61). The evolutionary history was in-
ferred with MEGA4 (59) using the neighbor-joining algorithm with p-distance
correction and pairwise deletion of gaps and missing data. A total of 100 boot-
strap replications were performed to test for branch robustness.
Gene expression studies. Harvest of P. reinekei MT1 cells and RNA extraction
were done as previously described (7). Reverse transcription (RT) and quanti-
tative real-time PCR were performed using a QuantiTect SYBR green RT-PCR
kit (Qiagen) for one-step RT-PCR in a Rotor-Gene 2000 real-time PCR ma-
chine (Corbett Research). Transcripts of ccaA, ccaB, ccaC, and ccaD were
quantified with the following primer pairs: CcaA-F (GGGCGCTTTCACACCA
ATGACC) and CcaA-R (GCAGGTGAGCGGGTCGGAAGTA), CcaB-F (GC
AGTTGAGGCGGCGGTTGTTA) and CcaB-R (GCTTGCCAACCAGGTCG
AATGC), CcaC-F (TGACACGTCCAAATCCCTGCCG) and CcaC-R (GCAA
GCGTGCGGCGTTATCAAT), and CcaD-F (GATGGCGTTGTCGGTCT
TGG) and CcaD-R (TGACGGTTTCAGGGCGGATA). A housekeeping
reference gene (ribosomal rpsL) was selected to normalize the results obtained
(9, 13). Real-time PCRs were carried out and relative expression ratios were
determined as previously described (7).
Mathematical calculations. Numerical calculations were performed with a
kinetic model built in SIMULINK v6.4.1 under the MATLAB v220.127.116.11 envi-
ronment (The MathWorks, Inc., Natick, MA) based on Michaelis-Menten ki-
netics using the kinetic constants experimentally determined here or previously
(7) and assuming a constant concentration of enzyme and zero-order kinetics for
oxygen and NADH.
Analytical methods. HPLC was performed as previously described (7).
Chemicals. 3-Chlorocatechol, 4-chlorocatechol, 3-methylcatechol, and 4-methyl-
catechol were obtained from Helix Biotech. 2-Methylmuconate, 3-methylmuconate,
and 3-chloro-cis,cis-muconate were freshly prepared from 3-methylcatechol, 4-meth-
ylcatechol, and 4-chlorocatechol, respectively, in 50 mM Tris-HCl, pH 7.5, 2 mM
MnCl2using chlorocatechol 1,2-dioxygenase TetC of Pseudomonas chlororaphis
RW71 (45) or partially purified C12OsalCfree of muconate cycloisomerizing activity.
cis-Dienelactone was kindly provided by Walter Reineke (Bergische Universita ¨t-
Gesamthochschule, Wuppertal, Germany) and Stefan Kaschabeck (TU Bergakad-
emie, Freiberg, Germany). Protoanemonin, 2-chloro-cis,cis-muconate, and trans-
dienelactone were prepared as previously described (4, 48).
Nucleotide sequence accession number. The nucleotide sequence reported in
this study was deposited in the DDBJ/EMBL/GenBank databases under acces-
sion number EF159980.
Characterization of a cycloisomerase transforming 3-chlo-
romuconate into both cis-dienelactone and protoanemonin.
Two MCIs, both transforming 3-chloromuconate into pro-
toanemonin, with minor quantities of cis-dienelactone, had
previously been characterized from P. reinekei MT1, and the
encoding genes had been localized (7). However, during
growth on 5-chlorosalicylate, the presence of a distinct enzyme
capable of transforming 3-chloromuconate was evident. This
enzyme, termed MCIccaB, eluted at 0.25 ? 0.04 M during HIC,
and as previously indicated (39), approximately equal amounts
of protoanemonin (50% ? 3%) and cis-dienelactone (47% ?
5%) were formed when proteins of such fractions were sup-
plemented with 3-chloromuconate. As the formation of such a
product mixture by any muconate or chloromuconate cyclo-
isomerase had not been previously observed, the enzyme was
purified to homogeneity. The native molecular mass of
MCIccaBwas estimated by gel filtration to be 350 ? 20 kDa,
and a single band of 43 ? 3 kDa was observed on SDS gels.
Thus, MCIccaB, like MCI of P. putida PRS2000 (22) or chlo-
romuconate cycloisomerase from Cupriavidus necator JMP 134
(23), may be a homo-octamer. N-terminal amino acid analysis
(SQGFVIGRVLAQRLDIPFSQPIRMSFGTLD) revealed no
significant similarity when these sequences were compared to
the sequences of other cycloisomerases available in databases.
muconate were transformed with high activity by this enzyme.
The highest turnover rate, 10-fold higher than with 3-methyl-
muconate, was observed with 3-chloromuconate (Table 1).
However, the specificity constants of 3-chloromuconate and
3-methylmuconate were almost equal, due to the significantly
higher Kmvalue with 3-chloromuconate. Activity of the en-
zyme with muconate was negligible, and at a substrate concen-
tration of 0.1 mM substrate, the activity was only 0.4% of that
with 3-chloromuconate. Thus, from the substrate utilization
profile, MCIccaBis clearly different from previously reported
MCIs, which are characterized by their high activity with mu-
conate (53, 54). It also differed from MCIsalCof MT1, which
has previously been characterized as being adapted for the
turnover of 3-methylmuconate (7) but retained a significant
activity with muconate. MCIccaBwas practically inactive with
2-chloromuconate, which is transformed at high rates by most
proteobacterial chloromuconate cycloisomerases described
thus far (31, 63, 64).
The fact that purified MCIccaBtransformed 3-chloromu-
conate stoichiometrically into equal amounts of protoanemo-
nin and cis-dienelactone contrasts with all previously described
cycloisomerases, which form either protoanemonin (MCIs) or
cis-dienelactone (chloromuconate cycloisomerases) as the pre-
dominant product (4, 39, 53, 54, 58). Following 3-chloromu-
VOL. 191, 20094-CHLOROCATECHOL DEGRADATION BY P. REINEKEI MT14907
conate transformation over time showed that both products
were formed at a constant ratio, indicating that the reaction
mechanism was independent of the substrate concentration.
It has previously been shown that trans-DLH of strain MT1
interferes with the cycloisomerization of 3-chloromuconate
catalyzed by MCIsalC(39), an enzyme encoded by the sal clus-
ter and induced during growth on chlorosalicylates (7), and it
was suggested that trans-DLH acts on intermediate 4-chloro-
muconolactone to form maleylacetate, thereby preventing pro-
toanemonin formation. To validate the notion that trans-DLH
can similarly interact with MCIccaB, 3-chloromuconate (0.12
mM) was transformed by enzyme mixtures comprising
MCIccaB(8.8 nM) and various amounts of trans-DLH (0.88 to
88 nM). As previously observed for MCIsalC(39), the simulta-
neous presence of trans-DLH decreased the amount of pro-
toanemonin formed (Fig. 1) but did not influence the extent of
cis-dienelactone formation, which was always 47% ? 5% of the
Characterization of a C12O specifically induced during
growth on 5-chlorosalicylate. As an MCI that was not encoded
by the previously described cat or sal gene cluster was induced
during growth on chlorosalicylate (7), we assessed whether a
distinct C12O was also induced under such conditions. In fact,
C12O activity was observed in protein fractions of cell extracts,
eluting at 0.23 ? 0.01 M NaCl during anionic-exchange chro-
matography, in addition to previously described C12OsalC,
eluting at 0.29 ? 0.01 M NaCl. HIC confirmed the presence of
a previously uncharacterized catechol dioxygenase, termed
C12OccaA, eluting at 0.52 ? 0.02 M (NH4)2SO4in 5-chloro-
C12OccaAwas purified to 95% purity by a two-step proce-
dure (see Materials and Methods). A prominent band of 30 ?
2 kDa observed after SDS-PAGE was subjected to N-terminal
sequencing. The determined N terminus (AVSRLAELVTAL
ESD) showed no significant similarity to any proteins in public
databases. It thus seems that C12OccaAis only distantly related
to previously characterized C12Os.
Kinetic data were measured directly in fractions comprising
C12OccaAwith a purity of at least 95% of the total protein.
Thus, it can be calculated that maximum turnover rates with
catechol of 2,375 U/g of protein correspond to activities of
2,500 ? 100 U/g C12OccaAand, based on a subunit molecular
mass of 29.424 kDa (as supposed for the predicted amino acid
sequence of C12OccaA[see below]), to a kcatvalue for catechol
of 1.2 ? 0.05 s?1(Table 1). This was approximately 1 order of
magnitude lower than those previously reported for C12OcatA
and C12OsalDand for other previously analyzed proteobacte-
rial C12Os (6, 37, 49, 51). A high turnover rate was observed
only for 4-methylcatechol, and a comparison of specificity con-
stants (kcat/Km) showed 4-methylcatechol to be the highly pre-
ferred substrate (Table 1). A similar substrate profile has so far
been observed only for C12OsalD, and it contrasts with that
reported for either catechol or chlorocatechol 1,2-dioxygen-
ases (3, 6, 11, 45). However, the degree of specificity of
C12OccaAwas even more remarkable than that of C12OsalD, as
specificity constants for 4-methylcatechol compared to those
for catechol, 4-chlorocatechol, and 3-methylcatechol differed
by factors of 30 to 100. Surprisingly, activity of C12OccaA
against 4-chlorocatechol was rather poor and was similar to
those of previously described C12Os (11, 30, 38, 51).
Characterization of the cca gene cluster. To localize genes
encoding C12OccaAand MCIccaB, degenerate primers based on
the N-terminal sequence were used for the amplification from
genomic DNA of a 72-bp DNA segment encoding part of
TABLE 1. Substrate specificities of C12OccaAand MCIccaBfrom P. reinekei MT1a
Activity with 0.1 mM
2.5 ? 0.1
0.24 ? 0.02
0.08 ? 0.01
24.0 ? 1.2
12.4 ? 0.3
2.0 ? 0.4
0.6 ? 0.1
0.6 ? 0.1
21.5 ? 2.5
1.2 ? 0.05
0.12 ? 0.01
11.5 ? 0.6
6.0 ? 0.15
0.55 ? 0.1
140 ? 10
26 ? 2.2
0.95 ? 0.1
105 ? 15
10.6 ? 1.2
40 ? 8
111 ? 8
11 ? 0.5
0.5 ? 0.05
aThe kinetic constants were determined as described in Materials and Methods. Standard deviations were calculated with the KaleidaGraph program. ND, not
FIG. 1. Ratio of maleylacetate and protoanemonin formed from
3-chloromuconate by mixtures of MCIccaB(8.8 nM) with various
amounts of trans-DLH (0 to 88 nM) of P. reinekei MT1. The reaction
mixtures contained 50 mM Tris-HCl, 2 mM MnCl2, pH 7.5, and 120
?M 3-chloromuconate. Substrate and product concentrations were
analyzed by HPLC.
4908 CA ´MARA ET AL. J. BACTERIOL.
MCIccaB. This allowed the design of a specific primer that,
together with a degenerate primer based on a conserved se-
quence motif identified in both proteobacterial muconate and
chloromuconate cycloisomerases, resulted in the amplification
of an ?400-bp DNA fragment. PCR-based screening of a fos-
mid library of the genome of strain MT1 using primers specific
for the gene encoding MCIccaBand that encoding trans-DLH
(8) showed that both genes were carried on the same fosmid,
which contained an approximately 37.6-kb DNA fragment
Sequencing of the insert revealed an approximately 5,100-bp
region with five open reading frames (ORFs) (Fig. 2) probably
involved in the degradation of aromatic compounds by strain
MT1. One ORF, designated ccaB, contained the above-iden-
tified 400-bp fragment encoding part of MCIccaBand can thus
be supposed to encode MCIccaB. The ccaB gene product
showed only moderate identity to proteobacterial MCIs (35%
to 42%), proteobacterial chloromuconate cycloisomerases
(33% to 40%), or muconate and chloromuconate cycloisomer-
ases (35% to 37%) identified in gram-positive microorganisms,
which in a phylogenetic analysis form separate branches with
low sequence identity to one another (Fig. 3). This indicated
that MCIccaBof strain MT1 forms a new branch, illustrating a
distinct evolutionary history. Upstream of ccaB, ccaA encoded
an enzyme with a deduced N-terminal sequence identical to
that of the above-characterized C12OccaAprotein. As observed
for MCIccaB, in a phylogenetic analysis, C12OccaAdoes not
cluster with any of the previously described separate branches
observed in intradiol dioxygenases (Fig. 3) and showed only
moderate identities with proteobacterial C12Os (30% to 38%),
proteobacterial chlorocatechol 1,2-dioxygenases (32% to
37%), or catechol and chlorocatechol 1,2-dioxygenases (31%
to 43%) from gram-positive microorganisms. Lower sequence
identity (27% to 33%) was observed with members of the
hydroxyquinol branch of intradiol dioxygenases (1, 17). The
predicted amino acid sequence of the ORF transcribed diver-
gently toward ccaA and designated ccaR showed up to 47%
sequence identity with identified and putative transcriptional
regulators of the IclR family, specifically with those of the
PobR subfamily of IclR-type regulators, comprising, among
others, proteins involved in the transcriptional regulation of
protocatechuate or 4-hydroxybenzoate degradative genes (62).
The highest sequence identity was observed with a putative
IclR regulator of Corynebacterium efficiensYS-314 (accession
number BAC19104); however, only slightly lower sequence
identity was observed with regulators with identified functions
(40% sequence identity with pcaR of P. putida PRS2000, in-
volved in regulation of protocatechuate degradation , and
39% sequence identity with pcaR of P. putida WCS358 ).
Downstream of ccaR, the previously described gene encod-
ing trans-DLH (8) and designated ccaC could be localized. The
deduced product of the downstream ccaD gene showed the
highest sequence homology with MARs, with the highest iden-
tity (59%) being observed with MAR TfdF2 of the 2,4-dichlo-
RT-PCR analysis of the cca cluster. The accumulation of
transcripts of ccaA, ccaB, ccaC, and ccaD was measured during
growth on 5-chlorosalicylate, salicylate, and acetate (noninduc-
ing negative control). When the relative expression levels be-
tween the target and the reference gene (rpsL) were compared
to those under noninducing conditions (at a ratio of 1), signif-
icantly higher levels of ccaA, ccaB, ccaC, and ccaD transcripts
were observed only in 5-chlorosalicylate-grown cells (50- to
150-fold) and not in salicylate-grown cells (Fig. 4).
Induction of C12OccaAand MCIccaBduring growth on
5-chlorosalicylate and 4-methylsalicylate. As two of the three
C12O-encoding catabolic gene clusters of strain MT1 (the sal
gene cluster and the cca gene cluster) were expressed during
growth on 5-chlorosalicylate, the importance of the encoded
C12Os and MCIs was assessed after growth on 5-chlorosalicy-
late and 4-methylsalicylate. Cell extracts were separated by
anionic-exchange chromatography, fractions were monitored
for transformation of 4-methylcatechol and 3-methylmu-
conate, and the activities were quantified. Both C12O and
muconate cycloisomerizing activities could be nearly quantita-
tively recovered (recovery was ?90% for C12O activity against
4-methylcatechol and 85 to 95% for MCI activity against
Fractions of cell extracts of 5-chlorosalicylate-grown cells
eluting at 0.23 ? 0.01 M NaCl and thus containing C12OccaA
accounted for only 20% ? 5% of the total activity against 0.1
mM 4-methylcatechol, whereas fractions eluting at 0.28 ? 0.02
M NaCl and corresponding to C12OsalDaccounted for 80% ?
5% of the total activity against 0.1 mM 4-methylcatechol (Fig.
5). Analysis of cell extracts from 4-methylsalicylate-grown cells
showed that only 7% ? 2% of the total activity against 4-meth-
ylcatechol was due to C12OccaA. Similar results were obtained
when activities against 0.1 mM 3-methylmuconate were ana-
lyzed, with only 7% ? 2% (cell extracts of 5-chlorosalicylate-
grown cells) and 4% ? 1% (cell extracts of 4-methylsalicylate-
grown cells) of the total activity due to MCIccaB. This indicated
that C12OccaAand MCIccaBwere of only minor importance
during the degradation of 4-methylsalicylate. In contrast, a
calculation of the respective activities against 0.1 mM 3-chlo-
romuconate indicated that 75% ? 5% of the total activity in
extracts of 5-chlorosalicylate-grown cells was due to induction
of MCIccaB, whereas C12OccaAseemed to be of minor impor-
tance for 4-chlorocatechol turnover (approximately 1% of the
total recovered activity against 0.1 mM 4-chlorocatechol). Cal-
culation of the metabolic flux of 0.1 mM 5-chlorosalicylate or
4-methylsalicylate in cells pregrown in each, based on the ki-
netic parameters obtained in this study or obtained previously
(7) (Fig. 5), supported the notion that 5-chlorosalicylate deg-
radation is driven predominantly by C12OsalDand MCIccaB
(95% and 81% of the overall flux in 5-chlorosalicylate-grown
cells, respectively) and that C120ccaBis of minor importance.
C12OsalDand MCIsalCwere of major importance for 4-meth-
ylsalicylate degradation (84% and 92% of the overall flux in
FIG. 2. Gene organization of a 5,129-bp region from P. reinekei
MT1 containing the cca gene cluster. The arrows indicate gene orien-
tations: ccaA, C12O gene; ccaB, MCI gene; ccaC, trans-DLH gene;
ccaD, putative MAR gene; and ccaR, putative transcriptional regulator
gene. The encoded enzymes are given below the gene clusters.
VOL. 191, 20094-CHLOROCATECHOL DEGRADATION BY P. REINEKEI MT14909
4-methylsalicylate-grown cells). It should be noted, however,
that the kinetic parameters used for these calculations reflect
their activities in the enzymatic test and not necessarily their
activities in situ.
Here, we report the identification of a set of five genes that
are located in a 5.1-kb region of the genome of P. reinekei MT1
and that encode enzymes involved in the degradation of
5-chlorosalicylate via 4-chlorocatechol (Fig. 6).
In addition to the ccaC gene, encoding trans-DLH (8), this
gene cluster comprised genes encoding functional C12OccaA
and MCIccaBproteins that were induced when the strain was
grown on 5-chlorosalicylate (but also on 4-methylsalicylate).
The presence of three distinct sets of (chloro)catechol 1,2-
dioxygenases and (chloro)muconate cycloisomerases raises the
question of their functions for the degradation of differently
substituted salicylates in strain MT1. On one hand, the induc-
tion of C12OccaAand MCIccaBduring growth on chlorosalicy-
late indicates their involvement in the degradation of chloro-
aromatics. On the other hand, C12OccaAwas found to be only
poorly active against 4-chlorocatechol, the central intermedi-
ate of chlorosalicylate degradation by MT1, and in its kinetic
properties against catechol and 4-chlorocatechol, this enzyme
resembles proteobacterial C12Os (6, 37, 49, 51). In contrast,
C12OsalD, being coinduced during growth on chlorosalicylate,
was reported to exhibit increased 4-chlorocatechol turnover
rates compared with other proteobacterial C12Os (7). In fact,
calculation of the relative activities against 4-chlorocatechol in
cell extracts and of the metabolic flux indicated that C12OsalD,
rather than C12OccaA, drives 4-chlorocatechol metabolism but
indicated some importance of C12OccaAfor 4-methylcatechol
The turnover of intermediate 4-chlorocatechol has been re-
ported to be a pathway bottleneck for the growth of strain MT1
on chlorosalicylates (42), and at higher chlorosalicylate loads,
4-chlorocatechol was shown to accumulate. As chlorinated cat-
echols are highly toxic to eukaryotic and bacterial cells (55),
the concomitant accumulation of 4-chlorocatechol results in
cell death and termination of degradative performance (43).
The induction of two C12Os may result in a more robust
degradative phenotype, avoiding to a significant extent the
accumulation of 4-chlorocatechol. Accordingly, Perez-Pantoja
et al. (43) showed that an efficient turnover of chlorocatechols
is essential for the growth of C. necator JMP134 on 3-chloro-
benzoate and that multiple copies of a chlorocatechol 1,2-
dioxygenase gene are necessary to efficiently deplete chloro-
catechols produced during 3-chlorobenzoate turnover by this
strain. Taking into account the low turnover rate of both
C12OsalDand C12OccaAfor 4-chlorocatechol, it can be rea-
soned that their combined actions are necessary for efficient
P. reinekei MT1 was originally isolated from a four-member
4-chlorosalicylate-degrading bacterial community in which two
other community members, namely, Achromobacter spanius
MT3 and Pseudomonas veronii MT4 (41), were supposed to
support degradation by depleting toxic metabolites, 4-chloro-
catechol and protoanemonin, formed by MT1 during chloro-
salicylate metabolism (42). Thus, it seems that MT1 is specif-
ically adapted to degrade chlorosalicylates in concert with
those strains due to rather ineffective chlorocatechol-trans-
forming enzymes that are not suited for highly effective min-
eralization of chlorosalicylates in pure culture (41).
As for ring cleavage activities, two muconate-cycloisomeriz-
ing activities were also induced during growth of MT1 on
chlorosalicylates. The major difference between these enzymes
is the fact that MCIsalCpredominantly catalyzes the formation
of protoanemonin, a reaction that trans-DLH can interfere
with to produce maleylacetate whereas MCIccaBcatalyzes the
transformation to approximately equal amounts of protoane-
monin and cis-dienelactone. As trans-DLH cannot interfere
with cis-dienelactone formation, MCIccaBcan ensure a rapid
metabolism of intermediate 3-chloromuconate but increases
the formation of the cis-dienelactone dead-end intermediate.
The presence of two MCIs assisting in the metabolism of
chlorosalicylates may equip MT1 with a certain level of meta-
bolic flexibility. Evidently, strain MT1 mineralizes 5-chloro-
salicylate through a complex metabolic interplay between en-
zymes encoded by the cca and sal gene clusters.
Specific inactivation of genes of the sal and cca gene clusters
will in future clarify their importance for the degradation of
FIG. 3. Dendrograms showing the relatedness of intradiol dioxygenases (A) and MCIs (B). The evolutionary history was inferred with MEGA4
(59) using the neighbor-joining algorithm with p-distance correction and pairwise deletion of gaps and missing data. A total of 100 bootstrap
replications were performed to test for branch robustness. The scale bars indicate amino acid differences per site.
FIG. 4. Relative expression levels of catabolic genes in salicylate-
and 5-chlorosalicylate-grown cells of P. reinekei MT1 as determined by
quantitative RT-PCR. The values represent n-fold change (mean of
triplicate samples) in the ratio of gene expression between the target
gene and the reference gene (rpsL) compared to expression under
noninducing conditions (for acetate-grown cells, this ratio was set at 1).
The error bars indicate standard deviations.
VOL. 191, 2009 4-CHLOROCATECHOL DEGRADATION BY P. REINEKEI MT14911
chlorosalicylates by strain MT1 and the effects exerted when
mutant MT1 strains have to interact with the above-described
Two other catabolic enzymes are encoded in the cca gene
cluster. The ccaC gene product (trans-DLH) has recently been
described as a zinc-dependent hydrolase (8) that interacts with
the cycloisomerization of 3-chloromuconate by hydrolyzing the
intermediate 4-chloromuconolactone to maleylacetate (Fig. 6).
The ccaD gene obviously encodes a MAR. Genes encoding
MARs have initially been observed in chlorocatechol gene
operons (28, 36, 56, 57), where the encoding enzymes catalyze
a crucial degradation step channeling the substrate into the
FIG. 5. Metabolism of 5-chlorosalicylate (A) or 4-methylsalicylate (B) by P. reinekei MT1. The kinetic constants of SalOH, C12OsalD, C12OccaA,
MCIsalC, and MCIccaBare indicated. The specific activity (U/g protein) was determined in cell extracts, and the contribution of each of the
(chloro)catechol 1,2-dioxygenases or (chloro)muconate cycloisomerases to the total activity against 0.1 mM 4-chlorocatechol or 0.1 mM 3-chlo-
romuconate (A) or against 0.1 mM 4-methylcatechol or 0.1 mM 3-methylmuconate (B) in 5-chlorosalicylate-grown (gray) or 4-methylsalicylate-
grown (boxed) cells was calculated after enzyme partial purification (given in percent and U/g protein). The enzyme concentrations (?mol/g
protein) in the cell extracts were calculated based on the kinetic parameters of the enzyme of interest. The contributions of isoenzymes to the total
metabolic flux of 0.1 mM 5-chlorosalicylate or 4-methylsalicylate by 5-chlorosalicylate-grown (gray) or 4-methylsalicylate-grown (boxed) cells were
calculated by MATLAB and are given in percentages in the arrows.
4912 CA ´MARA ET AL.J. BACTERIOL.
3-oxoadipate pathway (47). MARs are also involved in the
degradation of chloroaromatics via hydroxybenzoquinols, such
as in the degradation of 2,4,5-trichlorophenoxyacetate (25) or
2,4,6-trichlorophenol (34); in the degradation of sulfoaromat-
ics (16, 21); and in the degradation of natural aromatics, such
as resorcinol (9, 24).
The cca gene cluster of MT1 not only presents a novel gene
arrangement, but specifically comprises enzymes only distantly
related (C12OccaAand MCIccaB) or completely unrelated
(trans-DLH) to enzymes previously described as involved in
catechol or chlorocatechol metabolism. Also unexpected was
the observation of a gene encoding an IclR-type regulator
transcribed divergently compared to the ccaA and ccaB genes,
as catechol and chlorocatechol catabolic gene clusters are com-
monly under the control of a LysR-type regulator (62). Proto-
catechuate catabolic gene clusters, in contrast, are usually reg-
ulated by IclR-type regulators, such as PcaR of P. putida (50),
PcaU of Acinetobacter sp. strain ADP1 (18), PcaR of R. opacus
1CP (14), and PcaQ of Agrobacterium tumefaciens (40). A gene
organization similar to that in MT1 has so far been described
only by Eulberg and Schlo ¨mann (15) for the catABC gene
cluster from R. opacus 1CP, where a gene encoding an IclR-
type regulator is transcribed divergently to a gene encoding
C12O. However, in contrast to the observation by Eulberg and
Schlo ¨mann, who argued that after the divergence of the cat
genes found in Rhodococcus from other catechol genes the
original LysR-type regulator gene was replaced by one belong-
ing to the PobR subfamily of IclR-type regulators, no indica-
tions of the evolutionary events leading to the development of
the MT1 cca cluster can be given at this time, as both C12Occa
and MCIccaBseem to represent a new lineage in the phylogeny
of intradiol dioxygenases.
It is astonishing that despite the tremendous efforts in se-
quencing isolates and in isolating new organisms with new
catabolic properties, these new lineages have not yet been
observed. One of the possible reasons may be the restricted
substrate specificity for metabolism of specifically p-substituted
catechols and m-substituted muconates. Specifically, the cata-
bolic properties of MCIccaBdeserve special attention, as it
showed metabolic properties not yet reported for any cyclo-
isomerase, producing both cis-dienelactone (as do chloromu-
conate cycloisomerases) and protoanemonin (as do MCIs) (4,
39, 53, 54, 58). Studies of the mechanism of MCI have sug-
gested that the reaction proceeds via an enol/enolate to which
a proton is added to form muconolactone (19), as depicted in
Fig. 6. Similarly, the formation of protoanemonin from
3-chloro-cis,cis-muconate involves a protonation reaction,
whereas in the reaction of chloromuconate cycloisomerases
with 3-chloromuconate, the corresponding enol/enolate inter-
mediate is not protonated but rather loses the negative charge
by chloride abstraction (29). Replacement of Lys169 of P.
putida PRS2000 MCI, which is known to provide the proton for
the protonation reaction (19, 52), by alanine resulted in mu-
tants that were not able to form protoanemonin but rather
formed cis-dienelactone (29). However, as a protonating lysine
residue is also conserved in chloromuconate cycloisomerases,
as it is in MCIccaB, it was proposed that during the divergence
of chloromuconate cycloisomerases from MCIs the rate of
chloride elimination from the enol/enolate intermediate was
enhanced, even though residues that could accelerate chloride
elimination could not yet be identified in chloromuconate cy-
cloisomerases (29). MCIccaBappears from the mechanistic and
genetic points of view to be an evolutionary intermediate be-
tween chloromuconate cycloisomerases and MCIs, in which
the rate of dechlorination was enhanced compared to those of
MCIs (as was evident from the formation of cis-dienelactone)
but significant rates of proton addition were also observed (as
was evident from the formation of protoanemonin). Thus, a
detailed analysis of the substrate binding pocket of MCIccaB
could reveal important information about residues crucial for
The work was supported by the DFG-European Graduate College
We thank Rita Getzlaff (HZI) for N-terminal protein amino acid
sequencing. We gratefully acknowledge Iris Plumeier and Agnes Wal-
iczek for their excellent technical support and Melissa Wos-Oxley for
critical reading of the manuscript.
FIG. 6. Degradation of 5-chlorosalicylate by P. reinekei MT1. Designations of gene products are given below the reaction steps.
VOL. 191, 2009 4-CHLOROCATECHOL DEGRADATION BY P. REINEKEI MT1 4913
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