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Journal of Bacteriology, February 1999, p. 1309-1318, Vol. 181, No. 4
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Conversion of 3-Chlorocatechol by Various Catechol
2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol
Dioxygenase Region of Pseudomonas putida GJ31
Astrid E.
Mars,1
Jaap
Kingma,1
Stefan R.
Kaschabek,2
Walter
Reineke,2 and
Dick B.
Janssen1,*
Department of Biochemistry, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, 9747 AG Groningen, The Netherlands,1
and
Chemische Mikrobiologie, Bergische
Universität
Gesamthochschule Wuppertal, D-42097 Wuppertal,
Germany2
Received 20 July 1998/Accepted 3 December 1998
 |
ABSTRACT |
Pseudomonas putida GJ31 contains an unusual catechol
2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and
methylaromatics for growth. A 3.1-kb region of genomic DNA of strain
GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase
(cbzE) was cloned and sequenced. The cbzE gene
appeared to be plasmid localized and was found in a region that also
harbors genes encoding a transposase, a ferredoxin that was homologous
to XylT, an open reading frame with similarity to a protein of a
meta-cleavage pathway with unknown function, and a
2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to
catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol
dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol.
178:5930-5937, 1996). The substrate range and turnover capacity with
3-chlorocatechol were determined for CbzE and four related catechol
2,3-dioxygenases. The results showed that CbzE was the only enzyme that
could productively convert 3-chlorocatechol. Besides, CbzE was less
susceptible to inactivation by methylated catechols. Hybrid enzymes
that were made of CzbE and the catechol 2,3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide
inactivation and its substrate specificity were mainly determined by
the C-terminal region of the protein.
| |
INTRODUCTION |
Microbial degradation of most
chlorinated aromatics occurs via chlorocatechols as intermediates.
These chlorocatechols are usually further degraded via a modified
ortho-cleavage pathway. This pathway involves an intradiol
dioxygenase which cleaves the aromatic ring at the ortho
position. Dechlorination occurs further along the pathway
(43).
An alternative route for the degradation of catechol derivatives is the
so-called meta-cleavage pathway. This pathway is mostly involved in the degradation of methylated aromatics. It involves an
extradiol dioxygenase that cleaves the catechol ring at the 2,3 position, yielding a 2-hydroxymuconic semialdehyde derivative (47). Many extradiol dioxygenases have been cloned and
sequenced, and the crystal structures of two of them were determined
(11, 44). These and most other extradiol dioxygenases have a
two-domain structure, in which the C-terminal domain contains the
active site. The enzymes usually require Fe2+ as a cofactor
(9).
When extradiol dioxygenases are confronted with 3-chlorocatechol (3CC),
they usually become inactivated (e.g., see references 2, 5,
10, 13, 15, and 22). This inactivation
might be caused by the strong chelating activity of 3CC, which removes the Fe2+ cofactor of the enzyme (22), or by
suicide inactivation of the enzyme due to the formation of a reactive
intermediate or product, such as an acylchloride (5).
Inactivation of the catechol 2,3-dioxygenases results in a low turnover
capacity of the enzyme with 3CC that is insufficient for growth
(50).
It was assumed for a long time that it was impossible to metabolize
3-chlorinated catechols via the meta-cleavage pathway, because they would inactivate the extradiol dioxygenase. However, we
recently described a Pseudomonas putida strain that does
degrade chlorobenzene via 3CC by this pathway (19, 25). The
strain contains a novel chlorocatechol 2,3-dioxygenase (CbzE) that can efficiently cleave 3CC at the 2,3 position, leading to simultaneous ring cleavage and dechlorination. In this paper we describe the cloning
and sequence of the cbzE gene. Furthermore, we determined the substrate ranges and characteristics of 3CC conversion of several
catechol 2,3-dioxygenases and of constructed hybrid enzymes.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
P. putida GJ31 was
isolated on chlorobenzene; its characteristics were described
previously (25). Burkholderia cepacia G4 (32) contains a catechol 2,3-dioxygenase (TomB) that is
involved in the conversion of catechol and 3-methylcatechol (3MC)
(36, 45). Escherichia coli NM522(pWW15.3201)
contains a 2.1-kb EcoRI fragment carrying the gene for
C23OII of P. putida MT15 (20) in pUC18 and was a
gift from P. A. Williams (University of Wales, Bangor, Wales,
United Kingdom). Plasmid pTDN1-1018 contains a 2.1-kb
SstI-HindIII fragment from P. putida UCC2 in pHG327 on which tdnC is localized
(39) and was a gift from N. C. McClure (Flinders
University of South Australia, Adelaide, Australia). pAW31 was derived
from pEMBL9 and contains xylTE of P. putida mt-2
(3, 7).
E. coli JM101 (53) was used for cloning and
construction of hybrid dioxygenases, and E. coli BL21(DE3)
(48) was used to express catechol 2,3-dioxygenases that were
cloned behind a T7 promoter. pBluescript SK+ (Stratagene, La Jolla,
Calif.) was used as a cloning vector, and pGEF+ (made from pGELAF+ by
deleting dhlA [42]) was used to make
translational fusions of (hybrid) catechol 2,3-dioxygenase genes behind
a T7 promoter.
DNA isolation and hybridization and cloning of the chlorocatechol
2,3-dioxygenase gene.
Total DNA from P. putida GJ31 was
isolated by the method of Ausubel et al. (4). Plasmid DNA
was isolated from P. putida GJ31 by a modified method of
Kado and Liu (18) as described by Duetz et al.
(8). Southern hybridization and chemiluminescent detection
of plasmid DNA or genomic DNA that was digested with PstI
were done with, as the probe, a digoxigenin-labeled DNA fragment that
was generated by PCR according to the instructions of the manufacturer
of the kit (Boehringer, Mannheim, Germany). This fragment was obtained
by amplification of a DNA segment of P. putida GJ31 with
degenerated primers that were designed against the N-terminal amino
acid sequence
(SIMRVGHVSI NVMDMAAAVK HYENVLGLKT TMQDNAGNVY LKK) of CbzE
(19) (5'-AAXGTZATGGAXATGGC-3'; X = C/T, Z = G/A/T/C) and a conserved C-terminal region (YFFDP) of catechol 2,3-dioxygenases (5'-GGYTCYAAYAAYTA-3'; Y = A/G). A
PstI fragment of 3.1 kb of P. putida DNA that
hybridized with this probe was isolated from a 0.8% agarose gel and
ligated in pBluescript SK+, after which the ligation mixture was
transformed into electroporation-competent E. coli JM101
cells. Transformants were plated on Luria broth (LB) agar plates
containing 100 µg of ampicillin ml
1, 40 µg of
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside
ml1, and 0.4 mM
isopropyl--D-thiogalactopyranoside (IPTG). White colonies were screened for catechol 2,3-dioxygenase activity by spraying them with a 10 mM 4-chlorocatechol (4CC) solution. Positive colonies turned yellow owing to conversion of 4CC to a chlorinated 2-hydroxymuconic semialdehyde derivative.
Sequence analysis.
Cycle sequencing (29) was
performed on double-stranded DNA with the Amersham Thermo Sequenase
cycle sequencing kit with 7-deaza-dGTP and 5'-Cy5 fluorescent primers.
Sequence reactions were run on the Pharmacia ALF-Express automatic
sequencing machine (Amersham Pharmacia Biotech, Uppsala, Sweden).
DNA and protein databases were screened for homologous proteins with
the BLAST program (
1). Multiple sequence alignments
were
made in ClustalW of Lasergene for Windows (DNAstar, Inc.,
Madison,
Wisconsin), and the percent identity between two proteins
was
calculated by the program as 100 × consensus length/(consensus
length + mismatches + gaps). Putative ribosome binding sites were
identified by using the sequences of the 3' ends of 16S rRNA of
E. coli and
Pseudomonas aeruginosa
(
46).
Enzyme essays.
Crude cell extracts of B. cepacia G4, E. coli NM522(pWW15.3201),
E. coli JM101(pTDN1-1018), E. coli JM101(pAW31),
and E. coli JM101(pBScbzE) were used to measure catechol
2,3-dioxygenase activities of TomB, C23OII, TdnC, XylE, and CbzE,
respectively. Crude cell extracts were made from overnight cultures
that were grown on LB containing 100 µg of ampicillin
ml1 (LB-Amp) and 0.4 mM IPTG in the case of the
recombinant E. coli strains. B. cepacia G4 was
grown overnight on mineral medium (26) containing 20 mM
acetate and 2 mM phenol at 30°C. Two hours before the cells were
harvested, 2 mM phenol was added to ensure good induction of TomB
(45). Cells were harvested by centrifugation and washed with
ice-cold 0.1 M Tris-HCl (pH 7.5) containing 0.1 mM 1,4-dithiothreitol.
After cells were resuspended in a small volume of dithiothreitol,
they were disrupted by sonication and centrifuged for 30 min in an
Eppendorf centrifuge (10,000 × g, 4°C). A
fresh Fe(II)
(NH4)2(SO4)2 · 6H2O solution was added to a concentration of 0.1 mM to the
clear supernatant, after which the supernatant was used as a source of
crude cell extract. The protein content of the extract was determined
with Coomassie brilliant blue, with bovine serum albumin as a standard.
Catechol 2,3-dioxygenase activities in crude cell extract were measured
spectrophotometrically as described previously (
25)
with
different catechols at 0.1 mM. The conversion of catechol,
3MC,
4-methylcatechol (4MC), and 4CC to their corresponding
meta-cleavage
products was monitored at 375, 388, 382, and
379 nm, respectively,
with extinction coefficients of 36,000, 16,800, 31,500, and 39,600
liter mol
1 cm
1,
respectively (
40,
41). The
meta conversion of 3CC
to 2-hydroxymuconic
acid was monitored at 290 nm. The extinction
coefficient of the
product is 12,500 liter mol
1
cm
1 (
19).
The turnover capacity with 3CC was determined for several catechol
2,3-dioxygenases. It represents the amount of 3CC that
can be converted
per amount of enzyme (nanomoles per unit of activity
with catechol).
This was measured by adding a defined amount of
crude cell extract to
an assay mixture, which contained 115 to
140 µM 3CC in phosphate
buffer (
25), unless indicated otherwise.
The concentration
of 3CC was measured over time by reversed-phase
high-performance liquid
chromatography. For this, a Merck-Hitachi
L-6200 pump (Merck-Hitachi,
Darmstadt, Germany) was used that
was connected to a LiChrosorb RP18
column (Chrompack, Bergen op
Zoom, The Netherlands). The samples were
injected directly from
the incubation mixture with a Marathon-Basic
autosampler (Spark-Holland,
Emmen, The Netherlands) and eluted with
water containing 25% (vol/vol)
acetonitrile and 0.2% (wt/vol)
H
3PO
4. 3CC was detected at 210
nm with a
Merck-Hitachi L-4200 UV-visible light detector, while
acquisition of
the data was done with PC integration package software
(Kontron
Instruments, Milan,
Italy).
For most enzymes, the catechol 2,3-dioxygenase was rapidly inactivated
within the first one to two minutes of the incubations,
and
substrate-independent loss of enzyme activity was negligible
during
this short period. For these catechol 2,3-dioxygenases,
the number of
data points that could be obtained was insufficient
to be fitted to a
mathematical description of the depletion of
3CC, and the turnover
capacity with 3CC was calculated by dividing
the amount of 3CC that was
converted by the amount of enzyme that
was added via the crude cell
extract. The latter value was determined
as the amount of activity with
catechol that was present in the
extract.
When the conversion of 3CC continued for a longer period, such as with
CbzE, significant substrate-independent inactivation
occurred during
the incubation period. The spontaneous loss of
enzyme activity was
described with the equation
dE/
dt =
kE, and
substrate
conversion-dependent inactivation was described with
dE/
dt =
pdS/
dt. Provided that the concentration range of 3CC was
high
enough to allow full saturation of the enzyme throughout
the
conversion, the turnover of 3CC can be described by
dS/
dt =
VmaxE. Solving these differential equations
gives equation 1:
![S<SUB>t</SUB>=(S<SUB>0</SUB>k+S<SUB>0</SUB>pV<SUB><UP>max</UP></SUB>−V<SUB><UP>max</UP></SUB>E<SUB>0</SUB>+V<SUB><UP>max</UP></SUB>E<SUB>0</SUB>e<SUP>(<UP>−</UP>t · [k+p · V<SUB><UP>max</UP></SUB>])</SUP>)/(k+pV<SUB><UP>max</UP></SUB>)](/content/vol181/issue4/fulltext/1309/img001.gif)
|
(1)
|
In this equation,
St and
S0 are the concentrations (micromolar) of 3CC at
time
t and zero (minutes), respectively;
Vmax is
the dimensionless maximal specific
conversion rate of 3CC relative
to the rate with catechol;
E0 is the initial activity of the catechol
2,3-dioxygenase in the assay mixture (units of activity with catechol
per liter);
p is the coefficient that describes inactivation
of
the enzyme as a result of the conversion of 3CC, which is expressed
as the amount of enzyme activity that is inactivated per amount
of 3CC
that is converted (units per micromole); and
k is a
first-order
inactivation constant (minute
1) that
describes aspecific enzyme inactivation. Including the
latter constant
in the calculations was especially important for
CbzE and hybrids
thereof since CbzE is rather unstable, although
substrate-dependent
inactivation is relatively minor. To determine
the rate constant for
substrate-independent inactivation, an amount
of extract that was equal
to the amount used in the turnover measurements
was incubated in
phosphate buffer without 3CC. The remaining catechol
2,3-dioxygenase
activity was measured over time with catechol,
and
k was
obtained from the slope of the logarithmic plot of the
catechol
2,3-dioxygenase activity versus
time.
The values for
Vmax and
p were
estimated from the 3CC depletion curves by fitting to the equation with
the program Scientist
for Windows 2.0 (Micromath Scientific Software,
Salt Lake City,
Utah). The turnover capacity was calculated by
Vmax/(
k +
pVmax).
Protein expression and construction of hybrid catechol
2,3-dioxygenases.
The genes encoding CbzE and TdnC were amplified
with PCR by using primers against the start and end of the genes. The
N-terminal primers contained an NcoI site (P1,
5'-GCTGCTCCATGGGTATTATGAGAGTTGGC-3' for
cbzE; P2, 5'-GCTGCTCCATGGGTGTACTGAGAAT-3'
for tdnC; NcoI sites underlined). The
C-terminal primers contained a BamHI site (P3, 5'-GACGTCGGATCCTCATGTGTACACATC-3' for
cbzE; P4,
5'-GACGTCGGATCCTCAGGTATAGACGTC-3' for
tdnC; BamHI sites underlined, stop codons shown
in bold). To ensure high fidelity of the PCR product, Pwo
DNA polymerase or the Expand High Fidelity PCR system was used
according to instructions of the manufacturer (Boehringer, Mannheim,
Germany). Due to the introduction of an NcoI site in
cbzE, the second codon of cbzE was changed to
encode a glycine instead of a serine. The
NcoI-BamHI fragments were cloned between the
NcoI and BamHI sites of pGEF+.
Protein expression was done in
E. coli BL21(DE3). For this,
constructs of pGEF+ containing the desired genes were transformed
to
E. coli BL21(DE3) and the cells were grown overnight on
LB-Amp
agar plates at 30°C. The next day, the transformants were
collected
from the plates and used to inoculate 100 ml of LB-Amp. The
cultures
were grown at 15°C for 2 days, after which the cells were
harvested
and used to prepare crude cell extracts as described above.
The
amounts of soluble and insoluble catechol 2,3-dioxygenase that
were
produced by
E. coli BL21(DE3) were estimated by comparing
the protein profile of crude cell extract with the protein profile
of
whole cells on a 12.5% polyacrylamide gel containing sodium
dodecyl
sulfate (SDS) as described by Schanstra et al. (
42).
Insoluble protein aggregates (inclusion bodies) are absent from
crude
cell extracts because they are removed by centrifugation
(30 min,
10,000 ×
g, 4°C), while they remain visible in the
protein
profiles obtained with whole
cells.
Hybrid catechol 2,3-dioxygenases were made of CbzE and TdnC by using
PCR fusion reactions (
54). For this, fragments of
cbzE and
tdnC were amplified with PCR by using
primers that generated
overlapping regions. The fragments were fused
together by PCR
using P1 or P2 as the forward primer and P3 or P4 as
the reverse
primer. The sequences of the other primers that were used
were
as follows: P5,
5'-
GGTCGAGCCAGTGCGCCCCGGC-3'; P6,
5'-
GATCCAACCAGTGCGCGCCCGC-3';
P7,
5'-
GCGCACTGGTTGGATC-3'; P8,
5'-
GCGCACTGGCTCGACC-3';
P9,
5'-
GTCGCCCTGGGGGCCGACGGTCAA-3'; P10,
5'-
TGAGCCATCGGGGCCCACCAGCAC-3';
P11,
5'-
GGCCCCGATGGCTCA-3'; P12,
5'-
GGCCCCCAGGGCGAC-3';
P13,
5'-
TTCTTGGCCATGAC-3'; P14,
5'-
GTCATGGCCAAGAA-3'.
The nucleotides
of the primers that are complementary to
tdnC are shown in
italics, and the nucleotides that are complementary
to
cbzE
are underlined. This way, fusions were created between
amino acid
positions 147 and 148, 189 and 190, and 240 and 241
of CbzE and TdnC.
The PCR fusion products were cloned behind the
T7 promoter of pGEF+,
and the hybrid enzymes were expressed in
E. coli BL21(DE3).
The hybrid genes that were made with PCR fusions
were checked with
restriction analyses and cycle
sequencing.
Nucleotide sequence accession number.
The sequence obtained
in this study has been submitted to GenBank and is available under
accession no. AF109307.
| |
RESULTS AND DISCUSSION |
Cloning and sequencing of a genomic DNA fragment encoding a
chlorocatechol 2,3-dioxygenase.
The N-terminal amino acid sequence
of the chlorocatechol 2,3-dioxygenase (CbzE) of P. putida
GJ31 was determined previously and showed homology with other catechol
2,3-dioxygenases (19). This sequence was used together with
a conserved C-terminal sequence (YFFDP) from homologous catechol
2,3-dioxygenases to design degenerate oligonucleotide primers. A 761-bp
PCR product was obtained, the nucleotide sequence of which corresponded
with the N-terminal amino acid sequence of CbzE. This PCR product was
used as a probe in Southern hybridization experiments, and a 3.1-kb
PstI fragment of genomic DNA of P. putida GJ31
was found to hybridize with the probe. This fragment was cloned in
pBluescript SK+ to give pBScbzE.
The nucleotide sequence of the 3.1-kb fragment was determined (GenBank
accession no.
AF109307). A schematic representation
of the open reading
frames (ORFs) that were identified is given
in Fig.
1. The
cbzE gene starts at
position 1319 and encodes a
polypeptide of 314 amino acids with a
calculated molecular mass
of 34,951 Da, which corresponds to the
experimentally determined
mass of 33.4 kDa of CbzE (
19). The
N-terminal amino acid sequence
that was deduced from
cbzE
was identical to the N-terminal sequence
of 43 amino acids that was
determined with the purified CbzE protein
(
19), except for
the last amino acid. In front of
cbzE is a
putative ribosome
binding site (AGGAG).
View larger version (9K):
[in this window]
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FIG. 1.
A schematic representation of the organization of the
ORFs that were identified on the 3.1-kb PstI fragment. The
PstI cloning sites and a XhoI site that was
confirmed by digestion are shown. The positions of the start codons
that were identified are indicated, as well as the sizes of the three
complete ORFs. The position where a deletion of monooxygenase genes
that corresponded to tbmBCDE (17) may have
occurred is indicated with an arrow.
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|
Downstream of
cbzE, a fragment that encodes an ORF of 154 amino acids was found, which was preceded by a Shine-Dalgarno-like
sequence, GGA. This ORF (CbzX) was 41% identical to CmpX of
Sphingomonas sp. strain HV3 (GenBank accession no.
Z84817).
The
cmpX gene
is part of an operon encoding a
meta-cleavage pathway for catechol.
Its function is unknown
(
55).
Further downstream, the beginning of an ORF (CbzG) was found that is
similar to many 2-hydroxymuconic semialdehyde dehydrogenases,
of which
DmpC of
P. putida CF600 (GenBank accession no.
P19059)
(
35) was one of the most similar enzymes, having 56%
identity
with CbzG. These enzymes are responsible for oxidation of the
carbonyl group that is present in the
meta-cleavage products
that
are formed when catechols are converted by catechol
2,3-dioxygenases.
CbzG most likely has the same function. The
corresponding genes
are found in operons that encode
meta-cleavage pathways (e.g.,
see references
23,
52, and
55). The
cbzG gene
continues
until the
PstI cloning site and is preceded by a
putative ribosome
binding site (GAGAG) (Fig.
1).
Upstream of
cbzE, an ORF of 119 amino acids is localized
(CbzT), which is also behind a putative ribosome binding site (AAGG)
(Fig.
1). The derived amino acid sequence shows up to 40% identity
with chloroplast-type ferredoxins that are known to play a role
in
maintaining the iron ion of catechol 2,3-dioxygenases in the
reduced
(Fe
2+) state (
16,
37). The genes that encode
these ferredoxins
are found in front of the genes that encode catechol
2,3-dioxygenases
(e.g., see references
12,
23, and
52), and
cbzT probably
encodes such a
protein as
well.
At the beginning of the cloned fragment is a segment of an ORF (ORF1)
of 126 amino acids which has 61% identity with the C-terminal
part of
a transposase of
Pseudomonas pseudoalcaligenes JS45 (GenBank
accession no.
AF028594) (unpublished data). This strain grows
on
nitrobenzene and uses a
meta-cleavage dioxygenase for the
conversion
of the intermediate 2-aminophenol to 2-aminomuconic acid
semialdehyde
(
24). ORF1 continues until the
PstI
cloning site. Presumably,
the complete transposase gene is present in
P. putida GJ31, but
only the last part was
cloned.
In between ORF1 and
cbzT are two (incomplete) ORFs that are
homologous to components of toluene and phenol monooxygenases.
In front
of
cbzT is a fragment that starts at position 720 and
encodes a sequence (ORF3) of 91 amino acids which is up to 50%
identical to the C-terminal part of reductase components of these
monooxygenases like TbmF of
Pseudomonas sp. strain JS150
(GenBank
accession no.
L40033) (
17). TbmF itself consists of
355 amino
acids. This suggests that the N-terminal part of this gene
was
lost in
P. putida GJ31, also, because no suitable start
codon
or putative ribosome binding site was found. Immediately upstream
of ORF3 is an ORF (ORF2) the C-terminal part of which resembles
another
component of toluene and phenol monooxygenases. TbmA of
the
toluene/benzene-2-monooxygenase of
Pseudomonas sp. strain
JS150 (GenBank accession no.
L40033) (
17) is most similar
to
this part of ORF2, and it has a region of 23 amino acids (Pro-43
to
Val-65) that contains 16 amino acids that are identical to
the
corresponding region in ORF2. The region between Pro-39 and
Val-61 of
DmpK of the phenol monooxygenase complex of
Pseudomonas sp.
strain CF600 (GenBank accession no.
M37764) (
34) is 57%
identical to this region in ORF2. The ORF continues into the region
that encodes ORF1. The N-terminal part of ORF2 lacks any homology
with
TbmA or with any other protein in the database, and it is
unclear
whether there is a real start codon for ORF2. A GTG is
present at
position 385, which is immediately behind the stop
codon of ORF1 at
position 381, but no putative ribosome binding
site was found.
Alternatively, an ATG is present at position 328,
which is located
inside the fragment that encodes ORF1. The components
of the phenol and
toluene monooxygenases are normally encoded
by six genes that are
located in an operon the last gene of which
encodes the reductase
component. In front of this reductase gene
are the four genes that
encode the three subunits of the hydroxylase
component, and a small
subunit that increases the oxidation rate
of the aromatic substrate in
vitro. The first gene of these operons
encodes the TbmA-like component,
to which the C-terminal end of
ORF2 has similarity. The function of
this component is unknown
(
17,
33,
34). The presence of two
ORFs (ORF2 and ORF3) that
are similar to parts of TbmA and TbmF, which
are encoded by the
first and the last gene of the toluene monooxygenase
operon in
Pseudomonas sp. strain JS150 (
17),
suggests that recombinations
have occurred in this region of the genome
of
P. putida GJ31.
Strain GJ31 presumably does not contain
an intact toluene monooxygenase
since it most likely uses a dioxygenase
for growth on aromatic
compounds (
25).
The results show that the
cbzTEXG genes are clustered. The
genes that encode the enzymes of
meta-cleavage pathways are
usually
located in operons (e.g., see references
38
and
52), and the
other enzymes that belong to the
meta-cleavage pathway of
P. putida GJ31 might
also be located near
cbzTEXG, but they were not cloned
with
the 3.1-kb fragment. The presence of a part of a transposase-like
ORF
(ORF1) on the border of the cloned fragment could indicate
that the
meta-cleavage pathway genes of
P. putida GJ31 are
located
on some kind of transposable element, which has been observed
with other
meta-cleavage pathway operons as well
(
52).
Plasmid localization of cbzE.
Plasmid isolations
revealed that P. putida GJ31 contains a large plasmid which
hybridized with the probe against cbzE. When P. putida GJ31 was grown on benzoate, mutants arose that could no
longer grow on chlorobenzene and toluene but that were still able to
grow on benzoate and benzene by an ortho-cleavage pathway (25). The plasmid of the mutant (P. putida
GJ31M1) was slightly smaller than the wild-type plasmid and no longer
hybridized with the probe against cbzE (results not shown).
These observations indicate that the meta-cleavage pathway
genes are plasmid encoded.
Similarity of CbzE sequence to those of other catechol
2,3-dioxygenases.
ClustalW amino acid sequence alignments showed
that CbzE was most similar to the two-domain, iron-containing extradiol
dioxygenases that cleave monocyclic diols (9). On the basis
of phylogenetic analysis, Eltis and Bolin (9) proposed that
this family consisted of five subfamilies, and CbzE was most homologous
to the members of subfamily C. The identity of CbzE with the other
members of the subfamily ranged from 72% for TdnC of P. putida UCC2 (GenBank accession no. X59790) (unpublished data) and
a catechol 2,3-dioxygenase of B. cepacia AA1 (GenBank
accession no. U47111) (unpublished data) to 51% for TbuE of
Ralstonia pickettii PKO1 (GenBank accession no. U20258)
(23). The characterization of most extradiol dioxygenases
was not sufficient to attribute any biochemical or functional
properties to the various subfamilies (9), although it was
suggested that subfamily C might represent a group that has an
increased affinity for catechol and molecular oxygen (9, 23). However, this now seems unlikely since the recent
determination of the sequence of the catechol 2,3-dioxygenase gene of
B. cepacia G4 (tomB) (36) revealed
that the TomB enzyme also belongs to the 1.2.C subfamily, while
previous measurements showed that this enzyme does not have increased
affinities for oxygen and catechols (23).
An alignment of CbzE with the other members of subfamily C, as well as
XylE (GenBank accession no.
V01161) (
31) and BphC
(GenBank
accession no.
X66122) (
14), is given in Fig.
2.
XylE and BphC are 38 and 15%
identical to CbzE, respectively.
The crystal structure of the latter
enzyme is known (
11), and
the alignment of Fig.
2 is based
on the structure-validated alignment
that was made by Eltis and Bolin
(
9). The histidine and glutamate
residues that are known to
be involved in the binding of Fe
2+ in extradiol
dioxygenases are strictly conserved in all of the
enzymes.
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FIG. 2.
Structure-based amino acid sequence alignment of CbzE
with those of other extradiol dioxygenases. The secondary structure
elements are indicated below the sequence of BphC as open bars (for  helices) and arrows (for strands). The alignment was based on the
structure-validated alignment that was made by Eltis and Bolin
(9). Residues that are strictly conserved within the
catechol 2,3-dioxygenases are shown in boldface type, and the three
amino acids that are known to be involved in the binding of
Fe2+ are underlined. Sequences (GenBank accession numbers
in parentheses): CbzE, chlorocatechol 2,3-dioxygenase of P. putida GJ31; TdnC, 3-methylcatechol 2,3-dioxygenase of P. putida UCC2 (X59790) (unpublished data); C23O, Catechol
2,3-dioxygenase of B. cepacia AA1 (U47111) (unpublished
data); TomB, catechol 2,3-dioxygenase of B. cepacia G4
(36); Cdo2, catechol 2,3-dioxygenase II of P. putida MT15 (U01286) (unpublished data); TbuE, catechol
2,3-dioxygenase of R. pickettii PKO1 (U20258)
(23); XylE, catechol 2,3-dioxygenase of P. putida
mt-2 (V01161) (31); BphC, 2,3-dihydroxybiphenyl
1,2-dioxygenase of B. cepacia LB400 (X66122)
(14).
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Substrate range of several catechol 2,3-dioxygenases.
CbzE is
the only catechol 2,3-dioxygenase described so far that is able to
productively cleave 3CC at the 2,3 position. However, none of the
enzymes that belong to the same subfamily as CbzE has been tested for
the ability to convert 3CC. To find out whether this capacity was
restricted to CbzE or whether it was a feature which was shared with
the other members of the 1.2.C subfamily, we determined the substrate
ranges of some other members of this group. For this, the initial rates
of product formation were measured with various catechols for each
catechol 2,3-dioxygenase by using cell extracts. CbzE and XylE were
included as enzymes with good and very poor conversion of 3CC, respectively.
Large differences existed in the relative initial activities of the
various enzymes (Table
1). TdnC and
C23OII had the highest
activities with 3MC, which corresponds with the
data of McClure
and Venables (
27) and Keil et al.
(
20). TomB and XylE preferred
unsubstituted catechol,
although both enzymes occur in pathways
that have methylcatechols as
intermediates. Only CbzE was able
to convert 3CC at a high relative
rate and could sustain the conversion
of 3CC much longer than the other
enzymes. The relative activities
of the recombinant enzyme correspond
rather well with the relative
activities that were measured with CbzE
that was purified from
P. putida GJ31 (
19).
Significant conversion of 3CC with C23OII
and XylE was observed only
when much higher amounts of enzyme
were added. TdnC, TomB, and C23OII
became completely inactivated
within 20 s, while XylE was
inactivated more slowly than the other
catechol 2,3-dioxygenases of the
1.2.C subfamily (Fig.
3). The
plots of
the amounts of product formed in time yielded straight
lines when
catechol, 3MC, 4MC, or 4CC was converted by CbzE, while
a gradual
decrease in the product formation rate was observed
with the other
enzymes (Fig.
4).
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TABLE 1.
Activities and turnover capacities with 3CC of several
catechol 2,3-dioxygenases in crude cell extracts with
various catechols
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FIG. 3.
Specific production of 2-hydroxymuconate (2HM) by
several catechol 2,3-dioxygenases (C23Os) due to the conversion of 3CC
(100 µM). The production of 2HM was measured spectrophotometrically
at 290 nm. The amount of C23O that was added was determined as the
amount of C23O activity with catechol. Lines: , CbzE; · ···,
TdnC, ···, C23OII; , XylE.
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FIG. 4.
Formation of meta-cleavage products due to
conversion of catechol (), 3MC (···), and 4MC ( ) by CbzE
(A), TdnC (B), and XylE (C). The production of the
meta-cleavage compounds was measured spectrophotometrically
at 375, 388, and 382 nm for catechol, 3MC, and 4MC, respectively. For
CbzE, the product formation was linear in time with each catechol. For
TdnC and XylE, the rates of product formation with 3MC and 4MC
decreased over time due to inactivation of the enzyme.
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The measurements were done at catechol concentrations (100 µM) well
above the Michaelis-Menten constant (
Km) of the
catechol
2,3-dioxygenases since the
Km values
are generally below 10 µM
(
6,
19,
23,
49). This means that
the decrease in the
product formation rate cannot be caused by a
reduction of the
substrate concentration during the assays but is due
to some inactivation.
Harayama and coworkers already observed that XylE
was susceptible
to inactivation during conversion of 3MC and 4MC
(
6,
37),
which most likely results from the oxidation of the
Fe
2+ cofactor (
16,
37). Since this kind of
inactivation was not
observed for CbzE, this indicates that this enzyme
not only is
most resistant to inactivation by 3CC but also is able to
resist
inactivation by other
catechols.
Turnover capacity with 3CC of several catechol
2,3-dioxygenases.
The amounts of 3CC that can be converted by the
enzymes were measured (Table 1). The turnover capacity with 3CC of CbzE
was as much as two orders of magnitude higher than the turnover
capacities of TdnC, TomB, C23OII, and XylE, which shows that CbzE is
indeed very resistant to inactivation during conversion of 3CC. During these measurements the conversion of 3CC by TomB, TdnC, and C23OII stopped within 1 min after the enzyme and substrate were mixed. For
XylE, it took approximately 20 min before no further conversion took
place (Fig. 5). The turnover capacity of
XylE with 3CC was comparable to those with the other three enzymes.
This means that XylE, which belongs to the 2.A subfamily of class 1 extradiol dioxygenases (9), converted 3CC at a much lower
rate than the enzymes of the 2.C subfamily. Active XylE consists of
four identical subunits, and the tetramer has a
kcat of about 45,000 min1 with
catechol (30). With the turnover capacity of XylE of 64 nmol
of 3CC per unit of activity with catechol (Table 1), this means that
about 2,900 molecules of 3CC were converted per molecule of XylE
(tetramer) in the crude cell extract. This value is in the same order
of magnitude as the turnover of 1,000 that was previously determined
for XylE by using oxygen depletion measurements with 3CC
(50). For CbzE it took about 30 min before the enzyme stopped converting 3CC (Fig. 5).
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FIG. 5.
Conversion of 3CC by CbzE and XylE. The open circles and
squares show the concentrations of 3CC that were measured in the assay
mixtures with CbzE and XylE, respectively. The lines show the 3CC
depletion curves that were fitted through the data with equation 1.
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Both CbzE and XylE were inactivated when they were incubated in
phosphate buffer without 3CC. This was probably due to oxidation
of the
ferrous iron cofactor during the incubation. Because of
this, the
activities of extracts with CbzE and XylE were monitored
over time when
they were incubated in phosphate buffer in the
absence of 3CC. A
first-order decrease in the activities of the
enzymes was observed in
both cases with inactivation constants
(
k) of 0.028 min
1 (
r2 = 0.93) and 0.0098 min
1 (
r2 = 0.77) for CbzE and
XylE, respectively. Because of this, part
of the inactivation that was
observed during 3CC conversion by
both enzymes cannot be attributed to
3CC. The reciprocal of the
coefficient
p, which describes
the inactivation of the catechol
2,3-dioxygenase due to conversion of
3CC, would give the turnover
capacity of the enzymes when aspecific
inactivation is absent.
The value of
p is 0.033 U/µmol for
CbzE and 15.1 U/µmol for XylE,
which means that the turnover
capacities would be 44 and 3% higher
without aspecific enzyme
inactivation for CbzE and XylE,
respectively.
Expression of CbzE and TdnC in pGEF+.
Both cbzE and
tdnC were cloned behind the T7 promoter of pGEF+, to give
pGFcbzE and pGFtdnC, respectively. The plasmid was transformed into
E. coli BL21(DE3), which carries the T7 RNA polymerase gene
behind the lac promoter which can be induced with IPTG.
E. coli BL21(DE3) also expresses some T7 RNA polymerase
without IPTG (48). When the lac promoter was
induced with IPTG, a severe formation of inclusion bodies of CbzE and
TdnC was observed when whole cells were analyzed on SDS-polyacrylamide
gels. This did not occur in uninduced cells. Therefore, cells were
grown without IPTG, and this resulted in expression levels that were
comparable to that observed with pBScbzE in E. coli JM101
(Tables 1 and 2).
Upon cloning, the second amino acid of
cbzE was changed from
a serine to a glycine residue. The substrate range and turnover
capacity with 3CC of CbzE that was expressed from pGFcbzE were
somewhat different from the substrate range and turnover capacity
of CbzE that was expressed from pBScbzE (Tables
1 and
2). When
the
turnover measurements were repeated with a lower initial concentration
of 3CC (70 µM), the turnover capacity increased more than twice
to
18,000 nmol/U, which indicates that the enzyme is very sensitive
to
high concentrations of 3CC. The increase in turnover capacity
was
approximately one-third for CbzE that was expressed from pBScbzE,
which
is much less. Also, the enzyme that was expressed from pGFcbzE
was less
stable in phosphate buffer. The first-order inactivation
constants were
0.058 min
1 (
r2 = 0.99) and 0.028 min
1 (
r2 = 0.93) for CbzE
expressed from pGFcbzE and pBScbzE, respectively
(results not shown).
Besides the Ser-2-to-Gly-2 mutation, the
absence of CbzT in extracts
from
E. coli BL21(DE3)(pGFcbzE) could
be responsible for
this difference, since this protein is similar
to ferredoxins that are
involved in reactivation of catechol 2,3-dioxygenases
(
16,
37). Nevertheless, CbzE expressed from pGFcbzE had a
turnover
capacity with 3CC that was at least 35-fold larger than
the turnover
capacities of other catechol 2,3-dioxygenases, showing
that the
overexpressed enzyme is highly resistant to 3CC and that
the resistance
is due not only to CbzT or Gly-2. The substrate
range of TdnC that was
expressed from
E. coli BL21(DE3)(pGFtdnC)
was similar to
that of pTDN1-1018. The turnover capacity with
3CC was
smaller.
Substrate range and turnover capacity with 3CC of hybrid catechol
2,3-dioxygenases.
To find out which regions of CbzE and TdnC
determined the substrate ranges, a number of hybrid catechol
2,3-dioxygenases genes were made with PCR fusion and cloned into pGEF+.
The hybrids were designated H1 to H10 (Fig.
6).
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FIG. 6.
Schematic representation of the construction of hybrid
catechol 2,3-dioxygenases (H1 to H10). The primers (P1 to P14) that
were used in PCRs to generate DNA fragments that contained overlapping
regions for the PCR fusions are indicated with arrows.
 , polypeptide fragments derived from CbzE;
 , polypeptide fragments derived from TdnC. The amino acids of
CbzE and TdnC between which the fusions were made are indicated.
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One monomer of catechol 2,3-dioxygenase consists of two separate
domains that are thought to arise from a genetic duplication
(
9). The exchanges in hybrids H1 and H2 were made just in
front
of the start of domain 2, which is formed by the C-terminal half
of the protein and starts at
strand I (Fig.
2). The differences
in
the specific activities of the various (hybrid) enzymes that
were
measured in crude cell extracts (Table
2) were mainly due
to
differences in the concentration of soluble (hybrid) catechol
2,3-dioxygenase in these extracts, which was confirmed by analysis
on
SDS-polyacrylamide gels. Therefore, activities are given relative
to
the value found with catechol, which is well oxidized by both
TdnC and
CbzE.
Hybrids H1 and H2 yielded active enzymes. The substrate ranges and
turnover capacities with 3CC of H1 and H2 were comparable
to the
corresponding values of TdnC and CbzE, respectively (Tables
1 and
2).
Besides, the product formation plots with catechol,
3MC, 4MC, and 4CC
yielded straight lines for H2, which was also
observed for CbzE (Fig.
4), while the curved lines that were obtained
with H1 suggested that
this hybrid was inactivated during conversion
of these catechols in
manner similar to that of TdnC (results
not shown). These data indicate
that both the substrate specificity
and the susceptibility to
inactivation were determined by the
C-terminal domains of both enzymes.
The active site of catechol
2,3-dioxygenases is located in that domain
(
9).
Attempts were made to elucidate which amino acids mainly determined the
observed differences between both enzymes. This was
done by exchanging
smaller parts of the C-terminal domain, as
is depicted in Fig.
6.
The hybrids H3 and H4, in which the exchanges were made in the
beginning of the C-terminal domains, were normally active,
although a
large part of H3 that was produced was insoluble (Table
2). Compared
with TdnC, the replacement of amino acids 148 to
189 of TdnC with amino
acids of CbzE (H4) resulted in the loss
of the substrate preference for
3MC. This hybrid was even worsened
in its capacity to convert 3CC,
although it obtained a part of
CbzE. The complementary hybrid protein
(H3), in which the corresponding
fragment of CbzE was replaced by that
of TdnC, did not have an
improved relative rate for 3MC, while the rate
by which 3CC was
converted was reduced. Thus, the exchange of these
peptide segments
influenced the substrate specificity of the enzymes in
such way
that both hybrid enzymes lost their preference for substituted
catechols. The amount of 3CC that could be converted by CbzE dropped
significantly when amino acids 148 to 189 of CbzE were replaced
by
those of TdnC in H3. H4 also had a lowered turnover capacity
(Table
2).
When the middle parts of the C-terminal domains (amino acids 190 to
240) were exchanged in H5 and H6, no activity could be
detected in
crude cell extracts of
E. coli BL21(DE3) containing
the
constructed hybrid genes in pGEF+. This was also the case
when the
middle and last part of the domain (amino acids 190 to
314) were
exchanged together in H9 and H10. The presence of protein
inclusion
bodies in whole cells was apparent from SDS-polyacrylamide
gel
electrophoresis, which means that the hybrid catechol 2,3-dioxygenases
that were expressed were insoluble. Formation of inclusion bodies
is
thought to be caused by improper folding of enzymes (
28),
which indicates that the regions that were exchanged were not
similar
enough to yield properly folded hybrid
enzymes.
When the last regions (amino acids 241 to 314) were exchanged, both
hybrids H7 and H8 were soluble but had very low activities
(Table
2).
Therefore, the accuracy of the measurements of the
substrate range of
H7 and H8 was not very high. Besides, H7 was
very rapidly inactivated
when it converted methylated catechols.
H8, which was equal to TdnC
except for the last region, maintained
a high relative activity with
3MC, while it appeared to have an
improved relative rate for 3CC,
although the conversion stopped
within 30 s. The turnover capacity
measurement also suggested
that the ability to convert 3CC was improved
for H8 as compared
to the ability to convert TdnC. Besides, both
hybrids seemed to
have an improved relative rate with
4CC.
A mutant of XylE, in which a single amino acid in the last region of
the C-terminal domain was substituted (Val-291 to Ile-291),
had a
fourfold increase in the turnover capacity with 3CC and
other
substituted catechols, whereas the affinity for 3CC was
lowered
(
50). Like this mutant of XylE, CbzE has a valine at
the
corresponding position (Val-297) (Fig.
2), while all the other
catechol
2,3-dioxygenases have the isoleucine at that place. The
increased
turnover capacity with 3CC of H8 compared with TdnC
might be caused by
a similar effect of this substitution, and
this amino acid might play a
role in the resistance of CbzE to
inactivation with substituted
catechols.
None of the hybrids H3, H4, H7, and H8 had an improved resistance to
inactivation with the various catechols compared with
TdnC, since the
plots of the formation of product in time were
always curved (results
not
shown).
The properties of other hybrid catechol 2,3-dioxygenases have been
studied by Cerdan et al. (
6), who constructed several
hybrids of XylE and NahH. They concluded that the amino acid at
position 250 (His-250 in XylE, Gln-250 in NahH) mainly determined
the
sensitivity of the enzyme to inhibition with 3MC and the relative
kcat with this substrate. However, the amino
acids of the catechol
2,3-dioxygenases of the 1.2.C subfamily that
correspond to His-250
in XylE are always arginines (Arg-256), which
means that this
arginine cannot be responsible for the improved
resistance of
CbzE to inactivation with the various
catechols.
Hybrid catechol 2,3-dioxygenases were also made of XylE of the TOL
plasmid and XylE of
P. aeruginosa JI104 by using a
SalI
site that both corresponding genes had in common.
Although the
enzymes are 94% identical, the substrate range of the
former is
much more relaxed than that of the latter, which is highly
specific
for catechol (
21). The results indicated that the
last 43 C-terminal
amino acids probably determined the substrate
specificities, since
eight of the nine mutations that determined the
substrate range
of the hybrids are located in that region. The same
strategy was
used by Williams et al. (
51), who made hybrids
of two catechol
2,3-dioxygenases that were encoded by the TOL plasmid
pWW53. The
amino acid sequences of these enzymes are unknown, but these
researchers
also concluded that the C-terminal region determined the
binding
and catalytic specificity of the
enzymes.
Mechanism of toxicity and resistance.
Although our results
clearly showed that the substrate specificities and degrees of
resistance to inactivation are determined by the C-terminal domains of
catechol 2,3-dioxygenases, it remains to be established what mechanism
underlies the inactivation with the various catechols and which
biochemical properties are responsible for the resistance of the
CbzE enzyme. For 3CC, irreversible suicide inactivation was observed
with XylE (5), while Kle
ka and Gibson (22)
observed reversible inactivation of TodE of P. putida F1,
which was probably due to the chelation of the Fe2+
cofactor of TodE by 3CC. TodE is only distantly related to the catechol
2,3-dioxygenases used in this study (9), and its mechanism of inactivation seems to be different from that of XylE (5, 22).
When XylE was inactivated with 3CC, activity could not be restored by
addition of Fe
2+ and dithiothreitol (
5), which
suggests that the mechanism
of inactivation does not involve the
oxidation state or the loss
of the iron cofactor. Another possible
cause of the observed inactivation
is the nucleophilic attack of one or
more groups in the enzyme
on an electrophilic intermediate in the
reaction cycle. Preliminary
experiments with XylE showed that the
homodimeric protein band
of native enzyme migrated slightly faster on
SDS-polyacrylamide
gels than that of enzyme that was inactivated with
3CC, provided
that the samples were not boiled prior to application on
the gel.
Modification of XylE by 3CC also made the enzyme less
susceptible
to digestion with proteinase LysC. Complete digestion of
dialyzed,
inactivated XylE was possible only in the presence of 1 M
urea,
while native XylE could be digested in the absence of urea
(unpublished
results). These results might indicate that inactivation
by 3CC
involves a labile covalent modification which influences the
multimeric
form of XylE. The inactivation that was observed for XylE
and
NahH with methylated catechols (
6) was also related to
conversion
of the catechols and not to the catechols themselves since
the
inactivation constants were usually similar at 50 and 300 µM.
The increased resistance of CbzE to inactivation by both methyl- and
chlorocatechols might suggest that the mechanisms of
inactivation by
both compounds are similar. However, Hugo et al.
(
16) showed
that the conversion of 4MC by XylE resulted in the
oxidation of the
catalytic iron. The oxidized iron (Fe
3+) can be reduced by
XylT, which resulted in a reactivation of
the enzyme (
16,
37). The inactivation of XylE by methylated
catechols thus seems
to be a reversible process, in contrast to
the inactivation observed
with 3CC (
5). However, the EPR signal
of the oxidized iron
in XylE after inactivation by 4MC was clearly
distinct from the signal
of the catalytic iron that was aspecifically
oxidized by
H
2O
2 (
16). The authors suggested
that a putative
ligand that resulted from the conversion of 4MC
remained in the
active site, which might even hinder complete
reactivation by
XylT (
16). Perhaps such a ligand is also
formed during the conversion
of 3CC, which might bind in an
irreversible manner and prevent
the regeneration of the iron cofactor.
It should be noted that
the resistance of CbzE to 3CC is not
exclusively caused by a modified
interaction with CbzT, since increased
resistance was also found
with enzyme that was expressed from
E. coli in the absence of
CbzT, while purified CbzE also efficiently
converts 3CC (
19).
EPR studies of XylE that is inactivated with 3CC would provide more
insight in the mechanism that causes the inactivation.
This would also
help us to understand the biochemical basis for
the extraordinary
resistance of CbzE to methyl- and chlorocatechols.
If a specific group
in the enzyme is attacked by the electrophiles
that are formed during
the reaction with 3CC, the resistance of
CbzE to 3CC might result from
a modification of this target. Alternatively,
substitutions that lead
to a more rapid conversion of the toxic
intermediate could reduce the
lifetime of the reactive electrophiles,
which reduces the chance of
damage of the enzyme and increases
the resistance to
inactivation.
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ACKNOWLEDGMENTS |
This work was financed by a grant from the Dutch IOP
Environmental Biotechnology program and from the Deutsche
Forschungsgemeinschaft (Re 659/7-1).
We acknowledge P. Terpstra for the sequence analyses and U. Dehmel,
N. C. McClure, M. S. Shields, and P. A. Williams for
kindly providing the strains and plasmids with the catechol
2,3-dioxygenases.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Groningen Biomolecular Sciences and Biotechnology
Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen,
The Netherlands. Phone: 31-503634008. Fax: 31-503634165. E-mail: D.B.Janssen{at}chem.rug.nl.
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Journal of Bacteriology, February 1999, p. 1309-1318, Vol. 181, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
