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Journal of Bacteriology, September 1998, p. 4466-4474, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Similarities between the antABC-Encoded
Anthranilate Dioxygenase and the benABC-Encoded Benzoate
Dioxygenase of Acinetobacter sp. Strain ADP1
Becky M.
Bundy,
Alan L.
Campbell, and
Ellen L.
Neidle*
Department of Microbiology, University of
Georgia, Athens, Georgia 30602-2605
Received 4 May 1998/Accepted 18 June 1998
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ABSTRACT |
Acinetobacter sp. strain ADP1 can use benzoate or
anthranilate as a sole carbon source. These structurally
similar compounds are independently converted to catechol, allowing
further degradation to proceed via the
-ketoadipate pathway. In
this study, the first step in anthranilate catabolism was
characterized. A mutant unable to grow on
anthranilate, ACN26, was selected. The sequence of a
wild-type DNA fragment that restored growth revealed the
antABC genes, encoding 54-, 19-, and 39-kDa proteins,
respectively. The deduced AntABC sequences were homologous to those of
class IB multicomponent aromatic ring-dihydroxylating enzymes,
including the dioxygenase that initiates benzoate catabolism.
Expression of antABC in Escherichia coli, a
bacterium that normally does not degrade anthranilate,
enabled the conversion of anthranilate to catechol. Unlike
benzoate dioxygenase (BenABC), anthranilate dioxygenase
(AntABC) catalyzed catechol formation without requiring a
dehydrogenase. In Acinetobacter mutants,
benC substituted for antC during growth on
anthranilate, suggesting relatively broad substrate
specificity of the BenC reductase, which transfers electrons from NADH
to the terminal oxygenase. In contrast, the benAB genes did
not substitute for antAB. An antA point
mutation in ACN26 prevented anthranilate degradation, and
this mutation was independent of a mucK mutation in the
same strain that prevented exogenous muconate degradation.
Anthranilate induced expression of antA, although
no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity of
antABC did not prevent the use of
anthranilate as a sole carbon source. The
antABC genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation.
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INTRODUCTION |
Both the role of
anthranilate as an intermediary metabolite in tryptophan
degradation and the existence of bacteria able to use
anthranilate as a sole source of carbon and energy have
been known for many years (22). Nevertheless, the first step
in the aerobic degradation of anthranilate by bacteria
remains poorly characterized. Early attempts to purify the
multicomponent enzyme responsible for the initial oxidation step proved
to be unsuccessful. These attempts did, however, establish that
anthranilate was oxidized when two distinct
Pseudomonas protein fractions and Fe2+ were
present (26). Catechol was identified as the product of anthranilate dihydroxylation, and the corresponding
enzyme was shown to be a di- rather than a mono-oxygenase
(27, 46). No further information about this
anthranilate 1,2-dioxygenase (deaminating, decarboxylating; EC 1.14.12.1) has been reported in nearly 30 years.
Recently there has been renewed interest in microbial dioxygenases
(4, 6). This interest has stemmed in part from their importance in degrading a vast array of aromatic compounds in the
environment, in part from a fundamental concern about their biochemistry, and in part from their potential use in
bioremediation. Compounds similar in structure to
anthranilate, such as benzoate (Fig.
1) or 2-chlorobenzoate, are substrates of
multicomponent ring-hydroxylating dioxygenase systems (13, 18,
34). Several classes of these dioxygenases have been defined
based on the number of components, the types of iron-sulfur clusters
involved, and the types of flavin cofactors (4, 6). The
chromosomal benABC genes of the soil bacterium
Acinetobacter sp. strain ADP1 encode a class IB dioxygenase
that converts benzoate to a nonaromatic diol. Further catabolism of
this diol yields catechol, which is also the product of
anthranilate oxidation. The pathways for
anthranilate and benzoate degradation by strain ADP1,
therefore, are convergent (Fig. 1). By using a strategy based on
previous studies of benzoate and catechol degradation by ADP1 (14,
47), it was possible to select mutants unable to convert
anthranilate to catechol.

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FIG. 1.
Degradation of anthranilate and benzoate via
the -ketoadipate pathway. Relevant compounds, genes, and enzymes
are indicated.
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In this report, the characterization of one such ADP1-derived
anthranilate dioxygenase mutant is described. The
natural transformability of the Acinetobacter strains used
in these studies facilitated the identification of the wild-type
antABC genes, which restored growth of the mutant
with anthranilate as the sole carbon source. These
ant genes, shown to encode anthranilate
dioxygenase, were homologous in sequence to the benzoate
dioxygenase-encoding benABC genes. Comparisons between the
regulation and functions of the antABC genes and
those of their better-studied ben counterparts were
made. Whereas the formation of catechol from
anthranilate required only the dioxygenase-catalyzed step,
catechol formation from benzoate requires a second enzymatic step that
is catalyzed by the benD-encoded dehydrogenase (Fig. 1)
(33). Studies of the similar, but distinct,
ant- and ben-encoded multicomponent dioxygenases may reveal key features of enzyme substrate
specificity and catalytic efficiency.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Acinetobacter
strains are listed in Table 1.
Acinetobacter strains were derived from BD413, also
designated ADP1 (24). Taxonomic confusion has led to the
Acinetobacter calcoaceticus species designation for ADP1
being discontinued until further characterization is complete
(11a). Escherichia coli DH5
(Gibco BRL), S17-1
(43), and BL21(DE3) (44) were used as plasmid hosts. Bacteria were cultured in Luria-Bertani broth and minimal medium
at 37°C as previously described (39, 42). Carbon sources were added to minimal medium at the following final concentrations: 10 mM succinate, 3 mM benzoate, 3 mM 4-hydroxybenzoate, 3 mM
cis,cis-muconate, 2.5 mM anthranilate, or 2 mM
catechol. Antibiotics were added as needed at the following final
concentrations: tetracycline, 6 µg/ml; kanamycin, 20 µg/ml;
streptomycin, 25 µg/ml; spectinomycin, 25 µg/ml; and ampicillin,
150 µg/ml for Acinetobacter strains and 80 µg/ml for
E. coli.
DNA manipulation and plasmid construction.
Standard methods
were used for all chromosomal and plasmid DNA purifications,
restriction enzyme digestions, electrophoreses, ligations, and E. coli transformations (39). Plasmids are described in
Table 1 and Fig. 2. To isolate a
chromosomal ADP1 DNA fragment carrying the ant genes, a
recombinant plasmid library was constructed. Chromosomal ADP1 DNA
was digested with HindIII. Following electrophoretic separation, fragments of various size ranges were purified from a
0.7% agarose gel with the GeneClean purification kit (Bio101). A
fraction of DNA in the 4- to 6-kbp size range was ligated to the
cloning vector pZero2.1 (Invitrogen) and used to transform E. coli DH5
. This vector enables transformants with
recombinant plasmids to be selected on medium with kanamycin.
Plasmid pBAC103 (Fig. 2) was one of the recombinant plasmids
generated. Subclones of pBAC103 and pBAC163, whose isolation is
described below, were constructed by standard methods (39).

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FIG. 2.
Restriction map of the 6.5-kbp chromosomal
antABC region. The locations of genes, and their
transcriptional directions, are shown relative to some of the
restriction endonuclease recognition sites. Lines indicate the DNA
regions contained on recombinant pBAC plasmids. Plasmids shown in
boldface and marked by an asterisk were constructed with the pUC19
vector, and all others were constructed with the pZero2.1 vector. The
insertion sites of either omega or lacZ cassettes correspond
to those in strains and plasmids described in Table 1.
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To disrupt plasmid-borne genes, omega cassettes that can be excised
from pUI1637 or pUI1638 with one of several restriction
endonucleases
were used. The cassette from pUI1638 (

S) confers
resistance to
spectinomycin and streptomycin (
12,
37). That
from pUI1637
(

K) confers resistance to kanamycin (
12). Transcriptional
and translational stop signals follow each drug resistance
determinant.
In two plasmids, pBAC143 and pBAC112,
antC was disrupted by

S
(Table
1). In pBAC156, the
benC gene was disrupted by

K (Table
1). Open reading
frames (ORFs) near the
ant genes were disrupted
on
plasmids pBAC141(ORF2::

S), pBAC176 (ORF1::

K),
and pBAC244
(ORF3::

S) (Table
1). Plasmid pBAC162 was constructed
to generate
strains with chromosomal
antA::
lacZ transcriptional fusions (Table
1). A promoterless
lacZ cartridge that also confers
Km
r was used (
28). Plasmids pBAC214, pBAC226,
pBAC241, and pBAC147
(Table
1) were used to determine the mutation in
the
antA5024 allele as described below.
Acinetobacter transformation, strain construction,
and chromosomal mapping.
Naturally competent
Acinetobacter strains were transformed by linear DNA,
plasmid DNA, or crude DNA lysates as previously described (16, 23,
32). Some plasmids were introduced into Acinetobacter
recipients by conjugation from E. coli S17-1 (32, 43). Plasmids carrying modified regions of ADP1 DNA on replicons that are not stably maintained in Acinetobacter were used to
transform recipients, yielding new strains in which homologous
recombination had replaced the wild-type chromosomal allele with the
modified version. Strains ACN80 and ACN86 were constructed by this
method with the antC-disrupted plasmids pBAC112 and
pBAC143, respectively. The latter plasmid was used to generate ACN87
with ACN129 as the recipient strain. ACN103 was constructed by similar
methods with chromosomal disruptions of both antC and
benM (8). Strains constructed with ADP1 as the
recipient include ACN88 (by using pBAC141), ACN106 (by using pBAC156),
ACN107 (by using pBAC162), ACN130 (by using pBAC176), ACN169 (by using
pBAC194), and ACN204 (by using pBAC141). Strain ACN194 was generated by
using ACN107 as the recipient and pBAC244 as the donor DNA. This
plasmid was also the source of the disrupted ORF3 allele in ACN191.
Southern hybridization analyses confirmed that the mutated plasmid
alleles replaced the corresponding wild-type regions of the chromosome without integration of the entire plasmid.
In some cases, a mutation in one
Acinetobacter strain was
incorporated into the chromosome of a second strain by making a
crude
DNA lysate of the first strain and using it to transform
the second
strain (
16,
23). With this method, ACN115 was
generated
from ACN86 and ACN106, ACN118 was generated from ACN107
and ISA29,
ACN119 was generated from ACN107 and ISA25, ACN120 was
generated
from ACN107 and ACN88, and ACN170 was generated from
ACN169 and
ACN88. In the latter two strains, the
antA::
lacZ fusion of the
donor replaced
the deleted
antABC region of the recipient. Southern
hybridizations confirmed the expected chromosomal configurations.
To determine the locations of the
antABC genes on the
ADP1 chromosome, intact genomic DNA was prepared and digested as
previously
described (
16). The conditions for transverse
alternating-field
electrophoresis were the same as those of previous
studies (
16).
Isolation of chromosomal DNA in the region upstream of
antA.
In ACN88, the
S chromosomal insertion lies in
ORF2 and introduces an EcoRI recognition site not present in
the wild-type strain (Table 1 and Fig. 2). Chromosomal DNA from ACN88
was digested with EcoRI and ligated to
EcoRI-digested DNA of the cloning vector pUC19. Following
transformation of an E. coli host with the ligated DNA, a
single transformant with resistance to streptomycin and spectinomycin, characteristic of the ORF2::
S allele, was
obtained. This transformant harbored plasmid pBAC163 (Fig. 2), and
subsequent Southern hybridization and DNA sequence analyses confirmed
that pBAC163 carries DNA from the chromosomal region upstream of
antA.
Southern hybridization and DNA sequence analyses.
Southern
hybridization analyses were performed as previously described
(16). DNA probes were labeled with digoxigenin by random
priming, and probes were detected with antidigoxigenin-alkaline phosphatase conjugates and chemiluminescent substrates according to the
Genius System instructions (Boehringer Mannheim Corp.).
The DNA sequences of plasmids pBAC103 and pBAC163 and their subclones
(Fig.
2) were determined with double-stranded templates
and sequencing
primers, purchased from Promega, that recognize
the cloning vector. In
addition, 15 oligonucleotides, purchased
from Genosys Biotechnologies
Inc., were used as primers in sequencing
reactions (Table
2). In these reactions, oligonucleotides
1 to
4 were used with pBAC163 as the template DNA. The other
oligonucleotides
were used with pBAC103. An automated DNA sequencer
(ABI373A; Applied
Biosystems, Inc.) was used in the University of
Georgia Molecular
Genetics Instrumentation Facility. DNA sequences were
analyzed
with the Wisconsin Genetics Computer Group programs
(
11).
Determination of the sequence of the mutation in the
antA5024 allele.
Two different approaches were
used to identify the mutation(s) causing the temperature-sensitive
defect in AntA. In the first approach, the mutant allele was
amplified from purified chromosomal DNA by using oligonucleotides 5 and
10 (Table 2) as primers in a PCR with Pfu DNA
polymerase according to the instructions of the polymerase
supplier (Stratagene). Dimethyl sulfoxide was added to comprise 10% of
the total reaction volume. The PCR-amplified DNA product was gel
purified by using the QIAquick Gel Extraction kit (Qiagen) and digested
with ClaI and NsiI. This DNA was ligated to the
cloning vector pUC19 (49), which had been digested with AccI and PstI to form pBAC147. The sequence of
Acinetobacter DNA of pBAC147 was determined with primers
(from Promega) that recognized the pUC19 vector as described above.
In the second approach, gap repair methods (
17) were used to
isolate chromosomal DNA in the
antA region of ACN84.
With this
technique, a recipient strain is transformed with a
linearized
plasmid such that homologous recombination between
plasmid and
chromosomal DNA segments generates a circularized plasmid
in vivo.
The transforming plasmid is linearized to create a gap
between
two regions of homology, forcing recombination to occur
upstream
and downstream of the desired chromosomal
region, in this case
the mutant
antA DNA. The
recombinant plasmid generated in vivo
therefore will carry the
appropriate chromosomal DNA segment (
17).
Plasmid pBAC226
was digested with
ClaI to yield a linear fragment,
and this
DNA was used to transform ACN84. Homologous recombination
in vivo
between plasmid and chromosomal DNA sequences yielded
a circular
plasmid, pBAC241, carrying the
antA5024 allele. The
appropriate oligonucleotides (Table
2) were used for DNA sequence
determinations.
AntABC and
-galactosidase assays.
To measure
anthranilate dioxygenase activity, cell extracts were
prepared by sonication as previously described for other enzymes of the
-ketoadipate pathway (42). Anthranilate dioxygenase was
assayed spectrophotometrically by monitoring the decrease in
anthranilate concentration as indicated by
A310 (26). Protein concentrations
were determined by the method of Bradford (5), using bovine
serum albumin as the standard.
For

-galactosidase assays,
Acinetobacter cultures were
grown overnight in 5 ml of Luria-Bertani broth with or without the
addition of inducers, 3 mM benzoate, 3 mM
cis,cis-muconate, 2.5
mM anthranilate, or 2 mM
catechol. Cells were lysed with chloroform
and sodium dodecyl sulfate.
Following the removal of cell debris
by centrifugation, the

-galactosidase activity in the supernatant
fraction was
determined as described by Miller (
29).
Metabolite monitoring by HPLC.
For metabolite monitoring,
E. coli cultures (10 ml) were grown overnight on M9 medium
with glucose as a carbon source (39). Acinetobacter cultures were similarly grown on minimal
medium with either 10 mM succinate or 4 mM anthranilate as
the carbon source. Since ADP1(pBAC189) was unable to use
anthranilate as the sole carbon source, it was grown with
both 10 mM succinate and 2 mM anthranilate. When
appropriate, ampicillin was added for plasmid maintenance. Cultures
were diluted with 15 ml of the minimal medium used for overnight
growth, and in some cases, 250 µM
isopropyl-
-D-thiogalactopyranoside (IPTG) was added to
induce gene expression from the vector's lac promoter.
Monitoring was initiated after the addition of approximately 1 mM
anthranilate.
To monitor anthranilate metabolism, 1-ml culture samples
were centrifuged to pellet cells. Any cells remaining in the
supernatant
fractions were removed by passage through a
low-protein-binding,
0.22-µm-pore-size syringe filter (MSI). A
20-µl sample of the
filtrate was analyzed on a C
18
reversed-phase high-performance
liquid chromatography (HPLC) column
from Bio-Rad Laboratories.
Elution at a rate of 0.8 ml/min was carried
out with 30% acetonitrile
and 0.1% phosphoric acid, and the
eluant was monitored by UV detection
at 282 nm. Under these
conditions, the retention times for anthranilate,
catechol,
and
cis,cis-muconate were 6.0, 4.6, and 3.2 min,
respectively.
Peak areas corresponding to standards and experimental
samples
were calculated by using the ValuChrom software package
from Bio-Rad
Laboratories.
Nucleotide sequence accession number.
The sequence of the
6,529-bp antABC region was deposited in the GenBank
database (accession no. AF071556).
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RESULTS |
Isolation of a mutant, ACN26, unable to degrade
anthranilate.
The endogenous accumulation of
cis,cis-muconate (Fig. 1) is toxic (14).
Therefore, the presence of a compound that can be converted to this
metabolite prevents growth of strains in which the subsequent
metabolism of cis,cis-muconate is blocked, even when
an additional carbon source is available (14). Selection for
growth under these conditions can yield mutants that are unable to
form cis,cis-muconate. For example, the presence of benzoate in the growth media of catB deletion strains has selected
mutants that do not convert benzoate to cis,cis-muconate
(47). It seemed likely, therefore, that a similar strategy
could be used to isolate spontaneous mutants unable to
catalyze the initial hydroxylation of anthranilate.
Strain ADP205 (Table 1), which cannot degrade cis,cis-muconate because of a catMBC deletion,
was grown on solid medium with 3 mM 4-hydroxybenzoate as the carbon
source in the presence of 1 mM anthranilate, the potential
source of the toxic intermediate. Mutations preventing the
conversion of anthranilate to catechol, or those
preventing the conversion of catechol to cis,cis-muconate, should allow growth. Whereas
mutations of the first type were of interest, mutations of the second
type predominated as indicated by catechol production (data
not shown).
To identify the desired mutants,
anthranilate-tolerant strains were individually
transformed by the
catMBC genes that were
provided as linear
DNA from
XbaI-digested plasmid pIB1 (Table
1).
Previously described methods were used (
31,
32), and
transformants were selected with benzoate as the sole carbon source
to ensure repair of the chromosomal deletion. Under these conditions,
anthranilate dioxygenase mutants, but not catechol
dioxygenase
mutants, should grow, since catechol dioxygenase is
needed to
degrade benzoate (Fig.
1). Only one transformant
that could grow
on benzoate was obtained, ACN26 (derived from the
catM-catB deletion
parent ACN24). This strain was unable to
use either anthranilate
or
cis,cis-muconate as
the sole carbon source at 39°C. ACN26 could,
however, use
anthranilate as the sole carbon source at 25°C.
Different mutations affect cis,cis-muconate and
anthranilate metabolism.
ACN26 can degrade endogenous
cis,cis-muconate since it grows on benzoate. Mutations in
the mucK gene, encoding a transport protein, are known to
prevent growth on exogenous cis,cis-muconate (47). Therefore, the effect of introducing a wild-type
mucK gene into ACN26 was tested. ACN26 was transformed with
DNA from plasmid pADPW4, carrying mucK, and a resultant
strain was designated ACN84. ACN84 grew with
cis,cis-muconate as the sole carbon source, indicating that
ACN26 carries a defective mucK gene. ACN84 remained unable
to grow on anthranilate as the sole carbon source at
39°C. It is not clear why a mutation in mucK was isolated
in the selection for a strain unable to convert
anthranilate to cis,cis-muconate. Since similar
selections led to the isolation of strains with mutations in both
mucK and the structural genes encoding biodegradative enzymes (47), it may be that cis,cis-muconate
exported from adjacent cells under these isolation conditions
contributes to selective pressure for loss of the MucK uptake activity.
Isolation of the ant genes.
A fraction of 4- to 6-kbp HindIII-digested ADP1 DNA was able to
transform ACN26 to grow on anthranilate. Following ligation to a vector, this DNA was introduced into E. coli as
described in Materials and Methods. The recombinant plasmid-bearing
colonies were patched individually to solid anthranilate
medium onto which ACN26 cells had been spread. E. coli,
which does not use anthranilate as a carbon source, can
donate plasmid DNA in the natural transformation of ACN26. Plasmid DNA
able to repair the ACN26 mutation should allow growth. The
recombinant plasmids were not used in trans to
complement ACN26 directly, because the cloning vector is not stably
maintained in Acinetobacter, thereby making it difficult to retrieve DNA that restored growth by homologous recombination. Of
several hundred E. coli colonies screened, one carried a
plasmid, pBAC103, that restored growth of ACN26 on
anthranilate. This plasmid, with 4.9 kbp of ADP1 DNA, and
subclones derived from it were used for DNA sequence determination
(Fig. 2) (see Materials and Methods). Sequence analyses revealed
several ORFs (Fig. 2), three of which were designated
antABC based on their homology to a number of dioxygenase-encoding genes, described below.
Homology between AntABC and aromatic ring-hydroxylating
dioxygenases.
By database searches with the deduced amino acid
sequences, the 54-kDa AntA and 19-kDa AntB were found to be homologs of
the large and small subunits, respectively, of the terminal
oxygenases of class IB dioxygenases. The 39-kDa AntC resembled
the reductase components associated with these oxygenases. In pairwise
alignments of the Ant proteins with their counterparts, the percentage
of identical aligned residues ranged from 33 to 46% (Tables 3 to 5).
Enzymes most similar to AntABC included the highly specific benzoate dioxygenase and a toluate dioxygenase, with broad specificity, that dihydroxylates both benzoate and substituted benzoates. The former
is encoded by the ADP1 chromosomal benABC genes, and the latter is encoded by the Pseudomonas putida xylXYZ
genes of the TOL plasmid pWW0 (19, 34). Also homologous to
antABC were the cbdABC genes, isolated
from a conjugative plasmid of Burkholderia cepacia
(18). The CbdABC proteins catalyze the
dihydroxylation of 2-halobenzoates, yielding catechol.
Additional homologs of antA and
antB included the B. cepacia tftA and
tftB genes, which encode the large and small subunits of a
2,4,5-trichlorophenoxyacetic acid terminal oxygenase (10).
The proposed roles of AntABC are analogous to those of BenABC (Fig.
3).

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FIG. 3.
Proposed functions of the ant
gene-encoded proteins. By comparison with the functions of the
ben gene-encoded benzoate dioxygenase, indicated in the
boxes, the antAB genes encode the terminal dioxygenase
and benC encodes the reductase of a class IB dihydroxylating
multicomponent dioxygenase.
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Identification of the mutation that prevents growth on
anthranilate.
Plasmid pBAC116 (Fig. 2) transformed
ACN26 and ACN84 to grow with anthranilate at 39°C,
thereby localizing their mutations to a 298-bp region between the
ClaI and NsiI restriction recognition sites of
the antA gene. The corresponding region of ACN84 was isolated by gap repair methods (17), and DNA
sequence determination revealed a point mutation in the 43rd codon of
the antA structural gene that changed a T to an A
(position 2307 in the sequence under GenBank accession no.
AF071556). This same mutation was identified independently by PCR
amplification of the chromosomal region with Pfu DNA
polymerase (see Materials and Methods). As illustrated in Fig.
4, this mutation should cause a
conserved methionine residue to be replaced by a lysine in the
N-terminal region of the mutant protein.

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FIG. 4.
Alignment of dioxygenase protein sequences in the
N-terminal regions of the subunits. A substitution of K for M in
AntA causes a temperature-sensitive mutant enzyme that is
dysfunctional at high temperatures. Numbers indicate the amino acid
position of the adjacent residue. Residues identical to those of AntA
are shown in white on a black background.
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The antABC genes enable catechol to be formed from
anthranilate.
In E. coli, expression of the
antABC genes, on plasmid pBAC190 (Table 1), allowed
1 mM anthranilate to be converted to catechol in
approximately 4 h (Fig. 5A). A diol
intermediate was not detected by the HPLC monitoring techniques. An
isogenic strain carrying only the cloning vector did not metabolize
anthranilate under identical conditions (data not shown).

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FIG. 5.
Compounds in culture supernatant fractions. (A)
Anthranilate was converted to catechol by E. coli
BL21(DE3)(pBAC190) with the ADP1 antABC genes in
trans. (B) During anthranilate consumption (top)
by Acinetobacter strain ADP1 or by ADP1(pBAC189), with
the antABC genes in trans, catechol and/or
cis,cis-muconate accumulated (bottom). Prior to
anthranilate addition at time zero, the growth
substrate for ADP1 was anthranilate, whereas
ADP1(pBAC189) was grown with both anthranilate
and succinate. Concentrations were determined by HPLC methods.
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Surprisingly, the
antABC genes in
trans
on pBAC189 (Table
1) did not complement the inability of either ACN26
or ACN84 to
use anthranilate as a sole carbon source. In
addition, pBAC189
suppressed the ability of the wild-type
Acinetobacter strain ADP1
to use anthranilate as
the sole carbon source. ADP1(pBAC189) grew,
however, when succinate
was provided together with anthranilate.
It is possible
that succinate allows growth by reducing expression
of the
antABC genes compared to that when only
anthranilate is
available as the carbon source.
Consistent with this possibility,
ADP1(pBAC189) that was
grown with both compounds and then provided
with 1 mM
anthranilate removed all detectable
anthranilate from
the medium in approximately 4 h, a
rate slightly lower than that
of ADP1 that had been grown on
anthranilate (Fig.
5B).
During the initial phase of anthranilate catabolism by
ADP1(pBAC189), a small amount of catechol was detected,
approximately
30 µM at 1.5 h after anthranilate
addition (Fig.
5B, bottom).
In contrast, no catechol was detected
during anthranilate catabolism
by ADP1 that had initially
been grown with either anthranilate
(Fig.
5B, bottom) or
succinate (data not shown).
cis,cis-Muconate
was detected
during anthranilate catabolism by ADP1 and
ADP1(pBAC189)
(Fig.
5B, bottom). Since expression of the
antABC genes in
trans in
Acinetobacter led to the formation of a higher-than-normal
level of catechol during anthranilate catabolism, catechol
may
contribute to the inhibition of growth by ADP1(pBAC189) with
anthranilate
as the sole carbon source.
Anthranilate-mediated induction.
Anthranilate-grown ADP1
consumed anthranilate immediately upon its addition
to the culture, whereas with succinate-grown cells there was a delay of
approximately 2 h before consumption was detected (data not
shown). It appeared that the anthranilate degradation pathway, like similar pathways, was inducible. Consistent with this, anthranilate dioxygenase activity in cell extracts of
anthranilate-grown wild-type cells was approximately 0.03 µmol per min per mg of protein. In succinate-grown cells, the
activity was undetectable (less than 0.005 µmol per min per mg
of protein).
To characterize transcriptional-level regulation, strain ACN107,
in which the chromosomal
antA gene was replaced with an
antA::
lacZ transcriptional fusion (Fig.
2), was constructed.

-Galactosidase
(LacZ) activity was measured in
stationary-phase cultures. The
presence of
anthranilate in the growth medium of ACN107 increased
LacZ levels to 16,814 ± 2,337 Miller units, compared to 76 ± 47
Miller units in the absence of added inducers. To ensure that
induction resulted from anthranilate itself and
not a subsequent
metabolite, HPLC monitoring was used to confirm
that there was
no detectable catabolism of anthranilate by
the
antA-disrupted
ACN107 (data not shown).
Neither catechol nor
cis,cis-muconate
significantly
increased expression of the
antA::
lacZ
fusion relative
to that in uninduced cultures (data not shown).
Three ORFs near antABC are not required for
anthranilate degradation.
Since genes encoding
transcriptional activators are often adjacent to the targets of their
regulation, the DNA upstream of antA was isolated on
pBAC163 as described in Materials and Methods. The small, 417-bp ORF2
(Fig. 2) immediately upstream of antA was disrupted on
the chromosome of the wild-type strain and that of a strain with the
antA::lacZ fusion to generate strains
ACN204 and ACN120, respectively. Strain ACN204 was able to use
anthranilate as the sole carbon source. Moreover, the ORF2
disruption of ACN120 did not alter the ability of
anthranilate to induce expression of the lacZ
fusion. The presence of anthranilate in the growth medium
of ACN120 increased LacZ levels to 15,494 ± 1,781 Miller units,
compared to 64 ± 37 Miller units in the absence of added inducers. ORF2, therefore, did not appear to regulate transcription of
the antABC genes. In addition, disruption of ORF2 did
not have a cis-acting effect on transcriptional regulation.
ORF2 was not similar to sequences in the databases, whereas
homologs to ORF1 and ORF3, near
antABC (Fig.
2), were
detected.
Although the complete sequence of ORF1 has not been
determined,
its 5' region was homologous to those encoding
dehydrogenases
of a related family that includes PdxB, an enzyme that
oxidizes
4-phosphoerythronate in the biosynthetic pathway for
pyridoxine
(vitamin B
6) (
41). In an alignment of
147 amino acids of PdxB
with the ORF1-encoded peptide sequence, 47% of
the residues were
identical. Disruption of ORF1 on the chromosome of
strain ACN130
(Table
1) did not prevent the use of
anthranilate as a sole carbon
source.
The 5' region of ORF3, whose complete sequence has not been determined,
resembled genes in a family encoding AraC/XylS-type
transcriptional
activators (
15). One member of this family,
a putative
regulatory protein of
P. putida, was 40% identical
to the
ORF3-encoded peptide in a 76-amino-acid region (
37a).
In
ACN191 the chromosomal copy of ORF3 was disrupted (Table
1),
and, like
ACN130, ACN191 remained capable of growth with anthranilate
as the sole carbon source. In addition, expression of the chromosomal
antA::
lacZ transcriptional fusion
remained inducible by anthranilate
in strain ACN194, in
which the chromosomal copy of ORF3 was disrupted.
In ACN194, the fully
induced LacZ levels were 70% of those in
ACN107, indicating that ORF3
is not required for
antA expression.
Thus, specific
roles for ORF1, ORF2, and ORF3 in anthranilate
degradation
were not detected.
Localization of the ant genes on the ADP1
chromosome.
To map the ant genes by previously
described methods (16), a NotI recognition
sequence was introduced into the 3' end of antC by using
pBAC112 to replace the wild-type allele and generate ACN80 (Table 1).
Following the NotI digestion of genomic DNA and
electrophoretic separation by transverse alternating-field electrophoresis, the sizes of the six wild-type and seven ACN80 DNA
fragments were compared. In ACN80, the wild-type 1,090-kbp fragment was
replaced by two fragments, not present in the wild type, whose combined
sizes were equal to 1,090 kbp. A labeled probe made from DNA upstream
of antC between the ClaI sites in antA and antB (Fig. 2) hybridized to the
wild-type 1,090-kbp NotI fragment and to an 810-kbp
NotI fragment of ACN80. In addition, this probe hybridized
to a 490-kbp NotI fragment of a trpD mutant, ACN30, in which there is a NotI recognition site at map
position 3290 (hybridization data not shown). Collectively, these data located the antC gene at map position 3500 and indicated
its transcriptional direction to be clockwise, as depicted in Fig.
6.

View larger version (18K):
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|
FIG. 6.
Genome map of ADP1. The positions of genes are shown
relative to the six wild-type NotI recognition sites in the
chromosome. The chromosomal S insertion of ACN80 introduced a
NotI site at map position 3500, localizing the position of
antC as shown. The asterisk marks the position of the
hybridization probe indicating the direction of transcription as shown.
Clusters 1 and 2 mark the relative positions of large supraoperonic
regions containing numerous genes for aromatic compound degradation;
only one gene of each cluster is indicated.
|
|
Substitution of BenC for AntC.
Despite the
S insertion in
antC, strain ACN80 was able to grow with
anthranilate as the sole carbon source. A second
antC disruption strain, ACN86 (Fig. 2; Table 1), was
constructed and was also able to grow at the expense of
anthranilate. When antC was disrupted in
certain strains carrying mutations in the ben gene region,
however, the ability to grow with anthranilate as the sole
carbon source was lost. Specifically, combinations of the
antC::
S5086 allele with either the
benC::
K5106 allele (ACN115), the
(benB-benC5129) allele (ACN87), or the
benM::
K5008 allele (ACN103)
eliminated growth at the expense of anthranilate. The benM gene encodes a transcriptional activator of
benC expression (8). It appears, therefore, that
in the absence of antC, benC was required for
growth on anthranilate, most likely because the BenC
reductase can substitute for AntC in anthranilate
oxidation.
 |
DISCUSSION |
The isolation and analysis of the antABC genes
establish that anthranilate dioxygenase is evolutionarily
related to class IB dihydroxylating enzyme systems.
Dihydroxylating enzymes have been classified according to the
number and types of proteins in the multicomponent complex
(4, 6). Class I enzymes are comprised of a terminal
oxygenase and a flavin-containing NADH-dependent reductase. In
class IB enzymes the flavin cofactor is flavin adenine dinucleotide,
and the reductase contains a plant-type [2Fe-2S] cluster and a
presumed mononuclear iron center. The terminal dioxygenase contains a
Rieske-type [2Fe-2S] cluster. In anthranilate
dioxygenase, there are two subunits in the terminal oxygenase, the
larger of which, the
subunit, is presumed to contain both the
Rieske-type iron-sulfur and mononuclear iron centers.
In addition to the anthranilate dioxygenase-dependent route
for anthranilate catabolism (21), there
are at least two other evolutionarily distinct aerobic
pathways for degrading this compound. Some eukaryotic microbes
use a flavoprotein monooxygenase, anthranilate hydroxylase, to convert anthranilate to
2,3-dihydroxybenzoate (36). In addition, a novel pathway
for the aerobic degradation of anthranilate by a
denitrifying Pseudomonas strain, which has some
characteristics of both aerobic and anaerobic degradative routes,
has been described (1, 2).
Anthranilate dioxygenase and the homologous cbd-encoded
enzyme produce catechol from their substrates, whereas the
xyl- and ben-encoded dioxygenases yield
nonaromatic cis-diols that are converted to catechol in
subsequent NAD+-dependent dehydrogenase-mediated
steps. The direct formation of catechol most likely results from the
spontaneous decarboxylation and loss of the ortho
substituent when these steps are energetically favorable. No
cis-diol is detected during CbdABC-mediated
2-halobenzoate degradation (13, 18), and, similarly, none
was detected during anthranilate catabolism (Fig. 5). The
inference that the chemical nature of the substrate determines whether
a catechol or a cis-diol will form is consistent with
observations by Nakatsu et al. (30) that the requirement for
the CbaC dehydrogenase, following hydroxylation by the CbaAB
chlorobenzoate dioxygenase, depends on the substrate. In this example,
a cis-diol is formed when the substrate is 3-chlorobenzoate, but when the substrate is 3,4-dichlorobenzoate, elimination of HCl
occurs spontaneously, obviating the need for a dehydrogenase (30).
Comparisons of anthranilate and benzoate
dioxygenases.
The presence of benC allowed strains
lacking antC to degrade anthranilate,
suggesting that the BenC reductase was able to transfer electrons from
NADH to the AntAB terminal oxygenase component. Amino acids of class IB
reductases that are predicted to bind flavin adenine
dinucleotide, the [2Fe-2S] cluster, and those predicted to bind
NADH are conserved between AntC and BenC (6, 34). Amino
acids in these reductases that define substrate specificity, like
those involved in protein-protein contacts with the terminal oxygenases, have yet to be determined. Reductases, including BenC, can
donate electrons from NADH to artificial electron acceptors, demonstrating that the cognate oxygenase is not required for electron transfer. Moreover, electron transfer from several reductase components to a terminal oxygenase other than the authentic partner has previously been inferred (6, 10).
The ability of BenC to substitute for AntC requires the
ben
operon to be expressed in the presence of anthranilate.
Although
expression of this operon is inducible (
8), low
levels of constitutive
gene expression may allow some BenC to
facilitate the formation
of catechol which can then be converted to
cis,cis-muconate. Both
of these metabolites are known
inducers of BenM-activated
ben gene transcription
(
8). Consistent with this scenario,
benM disruption prevented
benC from substituting for
antC. Since the
benAB genes are cotranscribed
with
benC (
8), the inability
of
benA
to substitute for a defective
antA gene cannot be
attributable
to insufficient gene expression. Collectively, the results
indicate
that the substrate specificity of these dioxygenases is
determined
by the terminal oxygenase component.
Although the substrate specificity of the
Acinetobacter
benzoate dioxygenase has not been studied, that from
Pseudomonas
arvilla C-1 can hydroxylate anthranilate with 25%
activity relative to
benzoate as the substrate (
48). Whether
the substrate specificity
is determined by the

or

subunit, or
both, remains to be investigated.
An early study of toluate
dioxygenase, XylXY, implicated the smaller

subunit in substrate
specificity (
20). Recent studies of 2-nitrotoluene
2,3-dioxygenase, however, showed that the C-terminal region of
the

subunit defines this enzyme's specificity
(
35).
As expected, amino acids in the

subunits of the dioxygenases that
are predicted to coordinate iron-sulfur clusters, or those
that may
bind the mononuclear nonheme iron, are highly conserved
and are present
in AntA (
6,
34). The
antA mutation causing
anthranilate dioxygenase to be dysfunctional at 39°C, but
not
25°C, occurs in the N-terminal protein region, 12 residues from
a
conserved histidine likely to participate in binding the Rieske
[2Fe-2S] cluster (
34). The mutation was predicted to
replace
a conserved methionine with a lysine (Fig.
4), thereby
substituting
a positively charged residue for one that is hydrophobic.
The
significance of this substitution and its possible effect on
protein
folding at high temperature remain to be determined.
Expression and chromosomal organization of the
antABC genes.
The clustered antABC
genes may be cotranscribed, as are their ben counterparts
(8). The coding regions of antA and
antB are separated by only 2 nucleotides, and those of
antB and antC are separated by 11 nucleotides. Studies of a chromosomal
antA::lacZ transcriptional fusion,
described above, indicated that antA expression is
inducible by anthranilate itself and that the approximately 300-nucleotide region upstream of the antA
translational start signal is probably sufficient for
transcriptional control. Comparison of this region with that upstream
of benA did not reveal any obvious regulatory signals.
Studies of regulatory mutants suggested that neither of the
LysR-type activators known to control benzoate and catechol
degradation, BenM and CatM, controls ant gene
expression (7).
Anthranilate-to-catechol conversion appears to be tightly regulated,
since plasmid pBAC189, carrying the
antABC genes, does
not complement strains with the
antA5024 allele. The
plasmid-borne
antABC genes enable
E. coli to
convert anthranilate to catechol
(Fig.
5), and, therefore,
the lack of complementation in ACN84
and ACN26 may reflect toxic
metabolite imbalances during anthranilate
degradation by
strains with the
antABC genes in
trans. A
regulatory
problem is suggested, since pBAC189 suppresses the ability
of
ADP1 to use anthranilate as the sole carbon source. The
presence
of succinate together with anthranilate allows
ADP1(pBAC189) to
grow, but an unusually high level of catechol
accumulates from
anthranilate. Succinate may allow growth
of ADP1(pBAC189) by repressing
ant gene expression
and thereby limiting the levels to which catechol
accumulates. Although
previous studies indicate that the accumulation
of catechol, per se, is
not toxic at a concentration of 1 to 3
mM (
14), such high
levels could allow
cis,cis-muconate to reach
deleterious
levels. Feedback inhibition of anthranilate dioxygenase
by
catechol in
Acinetobacter strains might also play a role in
controlling metabolic flow.
The
antABC genes were not close to two supraoperonic
gene clusters involved in aromatic compound degradation. The
ben and
cat genes, involved in benzoate and
catechol degradation, respectively,
are grouped together in a region
greater than 20 kbp in length.
This region, cluster 2 (Fig.
6), is
separated from the
antABC genes by approximately
one-third of the ADP1 chromosome (
16).
In contrast, the
Pseudomonas aeruginosa ant loci map to a region
between
the
ben and
cat genes (
50,
51).
Although the
P. aeruginosa ant loci are involved in the
catabolism of anthranilate, individual
gene functions have
not been assigned, nor have these genes been
characterized
(
38). In ADP1,
antABC provide an unusual
example
of genes associated with the

-ketoadipate pathway that do
not
map either to the
ben-cat cluster or to a region, also
greater
than 20 kbp, that includes genes of the protocatechuate branch
of the pathway (cluster 1 in Fig.
6) (
16). The clustering of
many
Acinetobacter and
Pseudomonas catabolic
genes raises questions
not only about the evolutionary origin of these
regions but also
about their relationships with catabolic plasmids
(
19,
50,
51). Future studies are needed to determine whether
additional
genes involved in aromatic compound degradation are in the
vicinity
of the
antABC genes of ADP1.
 |
ACKNOWLEDGMENTS |
We gratefully acknowledge D. Matthew Eby for contributions to
plasmid construction, enzyme assays, and HPLC monitoring studies. We
thank Kim Gallagher, for genomic mapping and mutation localization, and
Ketan Patel, for the initial mutant isolation studies. We also
thank George Gaines, Don Kurtz, and Barny Whitman for helpful suggestions.
This research was supported by National Science Foundation grant
MCB-9507393.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Georgia, 527 Biological Sciences Building, Athens, GA 30602-2605. Phone: (706) 542-2852. Fax: (706) 542-2674. E-mail: eneidle{at}arches.uga.edu.
 |
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Journal of Bacteriology, September 1998, p. 4466-4474, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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