Cloning and functional analysis of aniline dioxygenase gene cluster, from Frateuria species ANA-18, that metabolizes aniline via an ortho-cleavage pathway of catechol.

Article abstract of DOI:10.1271/bbb.67.2351

Genes encoding an aniline dioxygenase of Frateuria sp. ANA-18, which metabolizes aniline via the ortho-cleavage pathway of catechol, were cloned and named tdn genes. The tdn genes were located on the chromosomal DNA of this bacterium and weren't clustered with catechol-degrading gene clusters. These results show that the ANA-18 aniline-degrading gene cluster is constructionally different from Pseudomonas tdn and Acinetobacter atd gene clusters, which degrade aniline via the meta-cleavage pathway of catechol and organize catechol-metabolic genes in the gene clusters. When cloned tdnQTA1A2B genes were expressed in Eschherichia coli, aniline dioxygenase activity was observed. Southern blot analysis revealed that homologues of the tdnA1A2B genes didn't exist in strain ANA-18. Disruption of the tdnA1A2 genes gave the parent strain ANA-18 a defect in aniline metabolism. On the basis of these results, we concluded that only the cloned tdn genes function as genes encoding aniline dioxygenase in strain ANA-18 although this bacterium had two catechol-degrading gene clusters.

Full text of DOI:10.1271/bbb.67.2351

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Cloning and Functional Analysis of Aniline
Dioxygenase Gene Cluster, from Frateuria Species
ANA-18, That Metabolizes Aniline via an ortho-
Cleavage Pathway of Catechol
Shuichiro MURAKAMI, Teruhiko HAYASHI, Tetsuya MAEDA, Shinji TAKENAKA
&
Kenji AOKI
To cite this article: Shuichiro MURAKAMI, Teruhiko HAYASHI, Tetsuya MAEDA, Shinji TAKENAKA
Kenji AOKI (2003) Cloning and Functional Analysis of Aniline Dioxygenase Gene Cluster, from
&
Frateuria Species ANA-18, That Metabolizes Aniline via an ortho-Cleavage Pathway of Catechol,
Bioscience, Biotechnology, and Biochemistry, 67:11, 2351-2358, DOI: 10.1271/bbb.67.2351
To link to this article: http://dx.doi.org/10.1271/bbb.67.2351
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Date: 05 November 2015, At: 13:16

Biosci. Biotechnol. Biochem., 67 (11), 2351–2358, 2003
Cloning and Functional Analysis of Aniline Dioxygenase Gene Cluster, from
Frateuria Species ANA-18, That Metabolizes Aniline via an ortho-Cleavage
Pathway of Catechol
Shuichiro MURAKAMI, Teruhiko HAYASHI, Tetsuya MAEDA, Shinji TAKENAKA, and Kenji AOKI†
Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture,
Kobe University, Nada, Kobe 657-8501, Japan
Received May 19, 2003; Accepted August 21, 2003
Genes encoding an aniline dioxygenase of Frateuria
sp. ANA-18, which metabolizes aniline via the ortho
UCC22 tdnA1, tdnA2, and tdnB genes encode the
-
large and small subunits of a terminal dioxygenase
and an electron transfer protein, respectively. TdnQ
and TdnT are related to the amino group transfer of
cleavage pathway of catechol, were cloned and named
tdn genes. The tdn genes were located on the chro-
mosomal DNA of this bacterium and weren't clustered
with catechol-degrading gene clusters. These results
show that the ANA-18 aniline-degrading gene cluster is
constructionally diŠerent from Pseudomonas tdn and
Acinetobacter atd gene clusters, which degrade aniline
via the meta-cleavage pathway of catechol and organize
catechol-metabolic genes in the gene clusters. When
cloned tdnQTA1A2B genes were expressed in Esch-
herichia coli, aniline dioxygenase activity was observed.
Southern blot analysis revealed that homologues of the
tdnA1A2B genes didn't exist in strain ANA-18. Disrup-
tion of the tdnA1A2 genes gave the parent strain
ANA-18 a defect in aniline metabolism. On the basis of
these results, we concluded that only the cloned tdn
genes function as genes encoding aniline dioxygenase in
strain ANA-18 although this bacterium had two
catechol-degrading gene clusters.
6
)
aniline and the release of ammonia.
Microorganisms metabolizing aniline via the
ortho-cleavage pathway are found in the genera
12)
1
0)
11)
Frateuria
,
Acinetobacter
,
and Rhodococcus.
Frateuria sp. ANA-18 is an excellent aniline-degrader
and metabolizes catechol by two catechol 1,2-diox-
1
3)
ygenases (CD) and cis,cis-muconate cycloisomerase
1
4)
isozymes
catechol. We cloned two catechol-degrading cat
cat gene clusters, consisting of diŠerent catABC
genes and genes encoding LysR-type regulators
after the conversion of aniline to
1
and
2
1
5)
(CatR
gene cluster was localized on a catabolic plasmid,
and was expressed at a lower concentration of an
inducer, cis cis-muconate, than the cat gene
1
and CatR
2
) from this bacterium. The cat
2
5)
1
,
1
1
6)
cluster. Although catechol-degrading genes have
been cloned in some aniline-degrading bacteria that
metabolize catechol via the ortho-cleavage path-
1
1,17–19)
Key words: tdn genes; aniline degradation; hydroxyla-
way,
genes encoding ADs have not yet been
tion; deamination; LysR-type regulator
cloned in these bacteria. In addition, there is great
interest in the number of gene clusters involved in
the conversion of aniline to catechol in Frateuria sp.
ANA-18 because two catechol-degrading gene
Aniline is converted to catechol through the
hydroxylation accomplishing deamination by
1
)
15)
microorganisms. Catechol is metabolized to organic
compounds involved in the TCA cycle via an ortho
or meta-cleavage pathway. Microorganisms
metabolizing aniline via the2meta-cleavage pathway
clusters were found in this strain.
-
In this paper, we report the cloning of genes encod-
ing AD from Frateuria sp. ANA-18. Southern blot
and gene disruption analyses revealed that the only
AD functioned in this bacterium.
–5)
are found in some genera.
Furthermore, genes
encoding aniline dioxygenases (AD), catalyzing the
conversion of aniline to catechol, have been cloned
Materials and Methods
6
)
from Pseudomonas putida UCC22, Acinetobacter
7
,8)
9)
sp. YAA,
and Pseudomonas sp. AW-2, which
Bacterial strains and bacterial culture. Frateuria
1
0)
metabolize aniline via the meta-cleavage pathway.
AD consisted of ˆve polypeptides encoded by
tdnQTA1A2B in P. putida UCC22. The strain
sp. ANA-18 was grown on 23 m
M
aniline or 22 m
C. For the 22
p-hydroxybenzoic acid medium, two solutions
M
p
m
-hydroxybenzoic acid medium at 30
9
M
†
To whom correspondence should be addressed. Fax:
81-78-882-0481; E-mail: kaoki kobe-u.ac.jp
＋ ＠
Abbreviations: AD, aniline dioxygenase; Ap, ampicillin; CD, catechol 1,2-dioxygenase; LB, Luria-Bertani medium; ORF, open reading
frame; Tc, tetracycline

2
352
S. MURAKAMI et al.
Fig. 1. Location of Cloned DNA Fragments and Genes.
Open arrows show ORFs and directions of their transcription. Probe 1 (a 1.8-kbp SacI fragment from p33D12) and probe 2 (a 1.0-kbp
Sal I fragment from p29C1) are shown by and , respectively. The left SacI and Sal I sites in probe 1 and probe 2, BanIII and Sac
I
sites used in the construction of the plasmid p29C11, and a Sal I site used in the construction of the plasmid p23FQ are located on the
vectors. A 1.0-kbp EcoRV-EcoRI and 1.2-kbp EcoRI-XhoI fragments used in Southern blot analysis of tdnA1A2 and tdnA2B as
probes are indicated by
and
, respectively. A solid triangle indicates a position where the CAT1 probe hybridizes. Arrows show
directions of the transcription from the lac promoter. Abbreviations; EI, EcoRI site; EV, EcoRV site; PI, PstI site; Pu, PvuII site; Sc,
r
SacI site; SI, Sal I site; XI, XhoI site; Tc , tetracycline resistance gene.
were prepared separately. Solution A contained 0.6 g
of K HPO , 0.3 g KCl and MgSO 7H O, 6 mg of
FeSO 7H O and CaCl 2H O, and 0.12 g of yeast
extract S (Nihon Seiyaku, Tokyo, Japan) in 600 ml
of water (pH 7.0). Solution B contained 2.7 g of
constructed from 9,600 transformants, as previously
17)
2
4
4
･
2
described. Recombinant plasmids of the transfor-
mants were ˆxed on a Hybond-N membrane
(Amersham Biosciences) according to the manufac-
turer's instructions. The CAT1 probe (5 -CATCCT-
TGAGCCTTCGCGCT-3 , corresponding to the
4
･
2
2
･
2
＋
p
-
?
hydroxybenzoic acid and 0.7 g of ammonium sulfate
in 300 ml (pH 7.0). Solution A was autoclaved, and
solution B was sterilized by ˆltration. The two
solutions were mixed at room temperature. When the
medium containing agar was prepared, solution A
containing 13 g agar was autoclaved and mixed with
?
sequence of a position of 58–77 bases in p50G12) was
labeled with T4 polynucleotide kinase (New England
32
Biolabs, Beverly, USA) and [g- P]ATP (ICN, Costa
Mesa, USA). Probe 1 (a 1.8-kbp SacI fragment from
p33D12), and probe 2 (a 1.0-kbp SalI fragment from
p29C1) (Fig. 1) were labeled with a Random primer
DNA labeling kit ver. 2 (Takara Bio, Otsu, Japan)
sterile solution B at 50 to 60 9C . Transformants
derived from strain ANA-18 were grown on 7.2 m
M
3
2
p
-hydroxybenzoic acid medium, prepared by the
and [
a
- P]dCTP (ICN). DNA fragments located
gene cluster were cloned by
addition of 0.9 g of
p
-hydroxybenzoic acid and
upstream of the cat
1
4
5 mg of yeast extract S in place of 2.7 g and 0.12 g
colony hybridization using the CAT1 probe. Probes
1 and 2 were used in colony hybridization to clone
DNA fragments ‰anking the inserts of the plasmids
p33D12 and p29C1, respectively. Colony hybridiza-
of these components in the 22 m
acid medium, supplemented with tetracycline (Tc)
6.25 ml). E. coli XL1-Blue was cultured in
Luria-Bertani medium (LB) at 37
supplemented with ampicillin (Ap) (100
12.5 ml), and kanamycin (50 ml).
M
p-hydroxybenzoic
(
mg
W
2
0)
20)
9
C, if necessary,
tion was done under standard conditions.
m
g
W
ml), Tc
The sequencing reactions were done by using a
Thermo sequenase primer cycle sequencing kit 7-
deaza dGTP (Amersham Biosciences) and plasmids
puriˆed with a FlexiPrep kit (Amersham Biosci-
ences). Reaction mixtures were run on a Shimadzu
DSQ-2000Lsequencer(Shimadzu,Kyoto,Japan).The
computer analyses of deduced amino acid sequences
were accomplished through the use of the BLAST
database searching program at the DNA Data Bank
of Japan. Identities of deduced amino acid sequences
were calculated by using a Genetyx-win software
(
m
g
W
mg
W
DNA manipulations, gene cloning, and sequenc-
ing. Standard methods were used for plasmid
DNA puriˆcations, restriction enzyme digestions,
2
0)
and E. coli transformations. Electroporation for
Frateuria sp. ANA-18 was done in the following
setting: 10 kV
experiments were done in pBluescript II vectors
Stratagene). A gene library of strain ANA-18 was
W
cm, 400 Q, and 25 mF. Subcloning
(

Aniline Dioxygenase Genes of Frateuria Species
2353
version 3 (Software Development, Tokyo, Japan).
pulsed cells were transferred into 7.2 m
M
p
-hydrox-
The DDBJ
W
EMBL
W
GenBank accession number for
ybenzoic acid medium, and cultivated at 30 9C for 8 h
the sequence reported in this paper is AB089795.
with shaking. Harvested cells were plated on the
.2 m -hydroxybenzoic acid medium supplement-
7
M
p
Plasmids construction. Plasmid DNA for gene
ed with Tc. Strain No. 49, grown on the plate, was
isolated as a single-crossover mutant. Strain No. 49
disruption was constructed in pBluescript II KS (
＋
)
(
D
PstI-SalI), carrying a multiple cloning site with
was subcultured on the 7.2 m
M
p-hydroxybenzoic
the deletion of a region between PstI and Sal I sites.
After digestion of the plasmid p29C1 with SmaI and
XbaI, a 5.1-kb fragment was recovered and ligated
acid medium ten times, and cultivated in 6 ml of the
same liquid medium. Cells harvested by centrifuga-
tion were spread on the same plates. Isolated colonies
were inoculated on an aniline plate supplemented
with Tc to examine their growth on aniline. Strains
No. 49-1, 49-2, and 49-3, showing a defect in the
aniline metabolism, were isolated as candidates for
double-crossover mutants with deleted tdnA1A2
genes, on the basis of the result that tdnA1A2
homologues didn't exist in strain ANA-18 (Fig. 2).
with SmaI and XbaI-digested pBluescript II KS (
＋
)
(
D
PstI-SalI). The resulting plasmid DNA was
r
named p29C1
was constructed by the substitution of a 1.2-kbp
EcoRV-EcoRI fragment in p29C1 with a 2.0-kbp
D. The plasmid pC1(tdnA1A2::Tc )
D
EcoRI-PvuII fragment containing a Tc resistance
gene from pBR322.
A 5.1-kb DNA fragment, obtained by the digestion
of p29C1 with BanIII and SacI, was ligated to a
Southern blot analyses. Homologues of the
tdnA1A2 genes and tdnA2B genes in strains No. 49,
49-1, 49-2, and 49-3, derived from strain ANA-18
by homologous recombination, were analyzed by
pBluescript II SK (
＋
) vector. A resulting plasmid
was named p29C11, in which, the tdnTA1A2B genes
were transcribed by the lac promoter. For expression
of the tdnQ gene, a 2.3-kbp SacI-SalI fragment
containing a tdnQ gene from p23F9 was ligated to
the low copy vector pMW218 (Nippon Gene, Osaka,
Japan) and the resulting plasmid was named p23FQ.
Southern hybridization. Each 1 mg of total DNA,
17)
puriˆed as previously described, was digested with
restriction endonucleases, and was ˆxed on a
Hybond-N
＋
membrane by a VacuGene XL vacuum
blotting system (Amersham Biosciences) after 1
z
Expression of tdn gene cluster in E. coli and AD
assay. E. coli XL1-Blue carrying various plasmid
DNAs was cultivated in 6 ml of LB medium
supplemented with antibiotics. When cell growth
agarose gel electrophoresis. A 1.0-kbp EcoRV-
EcoRI fragment containing partial tdnA1A2 genes
and a 1.2-kbp EcoRI-XhoI fragment containing
partial tdnA2B genes were labeled with an AlkPhos
direct labelling module (Amersham Biosciences), and
used as probes. The probes were hybridized with
DNA fragments ˆxed on the membrane according to
the manufacturer's instructions. DNA fragments
hybridized with the probes were detected using the
CDP-Star detection reagent (Amersham Biosciences)
according to the manufacturer's instructions.
reached early log phase, isopropyl-
topyranoside was added at a ˆnal concentration of
. After 5 h, cells were harvested and washed
with 0.8 sodium chloride solution. Washed cells
were mixed with 0.8 sodium chloride solution and
cell suspensions with the turbidity of 10 to 18 at
60 nm were prepared. Aniline-dependent oxygen
b
-
D
( )-thiogalac-
－
1
m
M
z
z
6
uptake showing AD activity was measured in a
Clark-type oxygen electrode (Yellow Springs Instru-
ment Co., Yellow Springs, USA) mounted in a water-
Induction of AD and CD. A parent strain,
Frateuria sp. ANA-18, and a strain defective in the
aniline metabolism, No. 49-1, were cultivated in
jacketed reaction vessel at 25
mixture consisted of 2.8 ml of 0.1
pH 8.0), 0.1 ml of 10 m aniline hydrochloride, and
.1 ml of the cell suspension. One unit of AD activity
was deˆned as the amount of enzyme that consumed
nmol of O per min.
9C . The reaction
M
Tris-HCl buŠer
900 ml of 22 and 7.2 m
media, respectively, for 20 h. Cells harvested by
centrifugation (13,000 , 10 min, 4 C) were washed
twice with 0.8 sodium chloride solution. Reaction
M
p-hydroxybenzoic acid
(
0
M
×
g
9
z
1
2
mixtures for the AD and CD induction and cell sus-
pensions used for enzyme assays were prepared as
1
4)
Isolation of double-crossover mutants deleting
tdnA1A2. Frateuria sp. ANA-18 cells, grown on
2 m -hydroxybenzoic acid medium, were
harvested from 10 ml of the culture by centrifuga-
tion, and mixed with 100 l of 10 glycerol after
washing with the same solution. The cells were
transformed by electroporation using 1.2 g of the
previously described.
cis cis-Muconate, added to the reaction mixture,
,
2
M
p
was enzymatically synthesized from catechol by the
catalysis of CD. The CD was prepared as previously
1
4)
m
z
described. A reaction solution consisted of 9.75 ml
of 0.1
solution (80 U). The conversion of catechol to cis
muconate started by the addition of 0.9 ml of 0.25
M
Tris-HCl buŠer (pH 8.0) and 12 ml of CD
m
,cis
-
r
plasmid pC1(tdnA1A2::Tc ) that causes the deletion
of tdnA1A2 genes by homologous recombination
M
catechol to the reaction solution, and was continued
at 30 C with shaking. The remaining catechol in the
(
Fig. 1). After incubation on ice for 10 min, the
9

2
354
S. MURAKAMI et al.
Table 1. Identity of Amino Acid Sequences Deduced from Cloned Genes
Identity (
88.4
z
)
Gene
tdnQ
Product or function
amino group transfer
Strain
References
ORF1
ORF2
ORF3
ORF4
P. putida UCC22
Acinetobacter sp. YAA
(6)
(7)
6
3.2
atdA1
component of aniline dioxygenase
62.0
tdnT
atdA2
amino group transfer
component of aniline dioxygenase
P. putida UCC22
Acinetobacter sp. YAA
(6)
(7)
3
7.7
83.4
tdnA1
atdA3
large subunit of aniline dioxygenase
P. putida UCC22
Acinetobacter sp. YAA
(6)
(7)
5
2.9
a
-subunit of aniline dioxygenase
79.0
tdnA2
atdA4
ORF1
small subunit of aniline dioxygenase
P. putida UCC22
Acinetobacter sp. YAA
Pseudomonas sp. AW-2
(6)
(7)
(9)
4
8
7.6
1.4
b
-subunit of aniline dioxygenase
small subunit of aniline dioxygenase
ORF5
ORF6
ORF7
75.7
tdnB
atdA5
ORF2
reductase component of aniline dioxygenase P. putida UCC22
reductase component of aniline dioxygenase Acinetobacter sp. YAA
reductase component of aniline dioxygenase Pseudomonas sp. AW-2
(6)
(7)
(9)
3
6
1.1
7.8
76.3
tdnR
atdR
ORF3
LysR-type regulator
LysR-type regulator
LysR-type regulator
P. putida UCC22
Acinetobacter sp. YAA
Pseudomonas sp. AW-2
(6)
(8)
(9)
3
7
7.5
4.0
37.0
7.0
ORF9
unknown
Alcaligenes eutrophus
SK4040
(23)
3
RSp0051 probable porin signal peptide protein
Ralstonia solanacearum accession No. (AL646076)
GMI1000 protein ID (CAD17202.1)
reaction solution was measured spectrophotometri-
p33D12 from a transformant showing a positive sig-
nal (Fig. 1). Preliminary sequence analysis revealed
that the insert DNA of p33D12 carried a tdnB
homologue, encoding a reductase component of AD
from P. putida UCC22. Colony hybridization using
probe 1 was done to select recombinant plasmids
with a region ‰anking the insert DNA of p33D12.
Two plasmids were isolated from two transformants
selected by the colony hybridization, and named
p29C1 and p33C5, respectively (Fig. 1). Further-
more, we screened transformants carrying recom-
binant plasmids hybridized with probe 2 and isolated
two plasmids, p8A7 and p23F9 (Fig. 1).
We found the complete 7 open reading frames
(ORFs) with the same orientation by sequencing
cloned fragments. ORF7 was located 175 bp down-
stream of ORF6. Intergenic regions between other
ORFs were less than 9 bp and ORF2 and 3, and
ORF3 and 4 were overlapped. The identities of the
amino acid sequences deduced from these ORFs are
summarized in Table 1. The amino acid sequences of
2
1)
cally using 4-aminoantipyrine. When catechol in
the reaction solution disappeared, the pH of the
reaction solution was adjusted to 8.0 with 1
hydroxide. By the addition of 0.9 ml of 0.25
catechol to the reaction solution, the synthesis of
cis cis-muconate was restarted. The cycle converting
catechol to cis cis-muconate, described above, was
repeated six times. After centrifugation (13,000
0 min, 4 C) of the reaction solution, the super-
natant was ˆltered by a Collodion bag (Sartorius
AG, Gottingen, Germany) to remove the enzyme.
N
sodium
6
)
M
,
,
×
g,
1
9
ä
The pH of the ˆltrate was adjusted to 2.0; the white
precipitate was collected by ˆltration, and then dis-
solved in ethyl acetate. Water was removed from the
sample by the addition of sodium sulfate and the
ethyl acetate layer was evaporated to dryness. The
white residue was analyzed by TLC as previously
1
)
described and identiˆed as cis
basis of the Rf values.
,cis-muconate on the
2
2)
CD activity was assayed as previously described.
One unit of CD activity was deˆned as the amount of
enzyme that converted 1 mol of catechol to cis cis-
the products of ORF1 to 6 showed more than 62.0
z
m
,
identities to those of AD components encoded by
6)
muconate per min. AD activity was assayed using cell
suspensions with the turbidity of 45 at 660 nm as
described above.
tdn genes from P. putida UCC22. Compared with
the UCC22 AD, the amino acid sequences of these
ORFs showed lower identities to those of AD encod-
7,8)
ed by atd genes from Acinetobacter sp. YAA. The
ORFs were organized as well as the alignments of the
tdn and atd genes. On the basis of these results, it
was concluded that ORF1 to 5 and ORF6 were
tdnQTA1A2B and tdnR, respectively, encoding AD
components and a LysR-type regulator. A product of
ORF7 consisted of 404 amino acid residues, and had
Results
Gene cloning and sequence analysis
To clone DNA fragments containing the upstream
region of the cat gene cluster, we screened the gene
1
library of Frateuria sp. ANA-18 by colony hybridiza-
tion using the CAT1 probe, and isolated the plasmid
z
37 identities to those of ORF9 from Alcaligenes

Aniline Dioxygenase Genes of Frateuria Species
Table 2. Expression of AD in E. coli Carrying Various Plasmids
2355
Plasmid
p29C11
AD activity (u Wm g dry cell)
･
0
0
0
p23FQ
p29C11
p29C11
＋
＋
pMW218
p23FQ
1
)
1.9
±
0.32
1
)
Values are the means
SD (n 4).
± ＝
2
3)
eutrophus SK4040,
and RSp0051, encoding a
probable porin signal peptide protein, from
Ralstonia solanacearum GMI1000 (accession no.,
AL646076; protein ID, CAD17202.1). An intergenic
region between ORF7 and catR was 382 bp, but
1
contained no ORFs. The amino acid sequences
deduced from a 1,959-bp sequence upstream of tdnQ
showed no identity to those of ORFs reported
previously.
Fig. 2. Southern Blot Analysis of tdnA1A2 in Frateuria sp.
ANA-18.
DNA fragments, digested with XhoI (lane 1), PstI (lane 2),
EcoRI (lane 3), EcoRV (lane 4), and EcoRI-EcoRV (lane 5),
were ˆxed on a membrane and hybridized with a 1.0-kbp
EcoRV-EcoRI fragment containing partial tdnA1A2 genes. M,
size marker.
Expression of AD genes in E. coli
AD activity was assayed in E. coli carrying plas-
mids with tdn genes (Table 2). No aniline-dependent
oxygen uptake showing AD activity was observed
in E. coli carrying p23FQ that contained only the
tdnQ gene or carrying the plasmid p29C11 with
tdnTA1A2B genes. On the other hand, E. coli
carrying tdnQTA1A2B genes showed the aniline-
dependent oxygen uptake, although the tdnQ gene
was located on a diŠerent plasmid from that of the
tdnTA1A2B genes, indicating that the catalysis of
strain No. 49, showing Tc resistance, was isolated as
a single crossover mutant. Furthermore, strains No.
49-1, 49-2, and 49-3, showing a defect in the aniline
metabolism, were isolated from strain No. 49 as
candidates for double-crossover mutants, which
deleted tdnA1A2 genes.
AD needs components encoded by tdnQTA1A2B
.
Deletion of tdnA1A2 genes in strains derived from
strain ANA-18 by homologous recombination was
examined by Southern blot analysis. When a 1.2-kbp
EcoRI-XhoI fragment containing tdnA2B was used
as a probe, the probe was hybridized with the only
Southern blot analysis of homologues of tdnA1A2
genes
Homologues of the tdnA1A2 genes were analyzed
by Southern blot analysis using partial tdnA1A2
genes as a probe. Figure 2 shows the results of the
Southern blot analysis. When the probe was
hybridized with the EcoRV-EcoRI-digested DNA
fragments from strain ANA-18, only the main band
appeared (lane 5). Although there were some extra
bands for digestion with other restriction en-
donucleases (lanes 1–4 for XhoI, PstI, EcoRI, and
EcoRV digestion), another band with a similar inten-
sity to each main band of 3.4-, 3.9-, 5.2-, and 4.2-kbp
in lanes 1–4 was not observed. These results suggest
that no signiˆcant homologues with high identity to
the cloned tdnA1A2 existed in this strain.
3.1-kbp SalI-Xho
I
fragment carrying partial
tdnTA1A2B genes in strain ANA-18 (lane 1 in
Fig. 3). This result shows that tdnB homologues
didn't exist in the strain in addition to tdnA1A2
homologues. In strain No. 49, a 1.9-kbp SalI-Xho
I
fragment containing partial tdnA2B genes on the
r
plasmid pC1(tdnA1A2::Tc ) (Fig. 1) appeared, in
addition to the 3.1-kbp band (lane 2), indicating that
r
pC1(tdnA1A2::Tc ) was introduced into the chro-
mosomal DNA of strain ANA-18 and two DNA
segments containing tdnA2B existed in the chro-
mosomal DNA of strain No. 49. On the other hand,
the 3.1-kbp band containing the original tdnTA1A2B
genes disappeared in strains defective in the aniline
metabolism, No. 49-1, 49-2, and 49-3 (lanes 3–5).
These results show that strains No. 49-1, 49-2, and
49-3 are double-crossover mutants with a deletion of
the tdnA1A2 genes.
Disruption of tdnA1A2 genes in Frateuria sp.
ANA-18
Disruption of tdnA1A2 genes was done to examine
how the cloned tdnA1A2 genes function as
components of aniline dioxygenase in Frateuria sp.
r
ANA-18. The plasmid pC1(tdnA1A2::Tc ) was
introduced into the chromosomal DNA of Frateuria
sp. ANA-18 by homologous recombination, and

2
356
S. MURAKAMI et al.
Fig. 3. Southern Blot Analysis of Strains Defective in Aniline
Metabolism.
Total DNA from Frateuria sp. ANA-18 (lane 1), strain No. 49
lane 2), strain No. 49-1 (lane 3), strain No. 49-2 (lane 4), and
(
Fig. 4. Induction of AD and CD with cis,cis-Muconate and
Aniline in the Parent Strain, Frateuria sp. ANA-18, and a Strain
Defective in the Aniline Metabolism, No. 49-1.
No. 49-3 (lane 5), were digested with Sal I-XhoI, and ˆxed on a
membrane. The ˆxed DNA fragments were hybridized with a
1
.2-kbp EcoRI-XhoI fragment containing partial tdnA2B genes.
Cells of strains ANA-18 and No. 49-1 were incubated in the
M, size marker.
reaction solution containing 10 m
and aniline (panel B), and AD and CD activities induced in the
cells were assayed at various times. Symbols: and , AD and
and ; AD
M
cis,cis-muconate (panel A)


CD activities in strain ANA-18, respectively.;
and CD activities in No. 49-1, respectively.


Induction of aniline-degrading enzymes
To conˆrm that the deletion of tdnA1A2 caused a
defect in aniline metabolism, the induction of AD
and CD was examined in the parent strain, Frateuria
sp. ANA-18, and a strain defective in the aniline
sequence showing similarity to that of the Pseudomo-
nas TdnT, which is involved in the amino group
transfer in the aniline degradation, is reported in the
analysis of amino terminal sequences of the aniline-
induced proteins in Acinetobacter lwo‹i K24
metabolizing aniline via the ortho-cleavage path-
metabolism, No. 49-1. By the induction with 10 m
M
cis cis-muconate, no production of AD was observed
,
in strain ANA-18 and No. 49-1; CD, on the contrary,
was produced in both strains (panel A in Fig. 4).
These results show that catechol-degrading enzymes
are functional in both strains. Panel B shows the in-
2
4)
way. However, this is the ˆrst report describing
the cloning of aniline-degrading tdn genes from
bacteria metabolizing aniline via the ortho-cleavage
pathway.
duction of AD and CD with 10 m
M
aniline in both
strains. The AD was induced by incubation for 30 h
in strain ANA-18, and increased after 36 h. The
production of CD was also induced by incubation for
The deduced amino acid sequences of
tdnQTA1A2BR cloned from Frateuria sp. ANA-18
showed more than 62.0 identities to those of the
z
AD components found in P. putida UCC22, which
metabolizes aniline via the meta-cleavage pathway
(Table 1). Furthermore, we found a similar sequence
to the UCC22 tdnQ promoter region containing
3
0 h because cis
,
cis-muconate, an inducer of the ex-
1
4)
pression of catechol-degrading gene clusters, was
generated from aniline by induced AD. On the other
hand, no induction of either enzyme was observed in
strain No. 49-1. These observations show that the
defect in the aniline metabolism in this strain was
caused by the loss of ability for the production of
AD.
possible
35 and 10 sequences and inverted
－ －
repeats where a regulator in the tdn gene cluster,
6
)
TdnR, may be bound, upstream of the ANA-18
tdnQ gene (Fig. 5). In addition to the identities
between ANA-18 and UCC22 ADs (Table 1), the
similarity of nucleotide sequences in the tdnQ
promoter regions suggests that these tdn genes were
derived from a common ancestor. Interestingly, T-11
nt-A sequences, reported as a binding motif of LysR-
Discussion
We found 7 ORFs in a ‰anking region of the DNA
fragment inserted in p50G12 (Fig. 1), and concluded
that the 6 ORFs of them encode AD and a LysR-type
regulator on the basis of their high identities to
reported ADs (Table 1) and the same organization as
tdn and atd gene clusters encoding ADs. In a recent
2
5)
type regulators, weren't found in the inverted
repeats of both strains and there may be unknown
binding motifs of LysR-type regulators in the tdnQ
promoter regions. The aniline-degrading genes of
study, a protein with an NH -terminal amino acid
2

Aniline Dioxygenase Genes of Frateuria Species
2357
Fig. 5. Alignment of tdn Promoter Regions from Frateuria sp. ANA-18 and P. putida UCC22.
Identical nucleotides are indicated by asterisks. The transcriptional initiation site for tdnQ and putative
－
35 and
－
10 sequences in
6)
6)
the strain UCC 22 sequence are indicated by
＋
1 and dotted lines, respectively. Inverted repeat sequences in strain UCC22 and
ANA-18 are underlined and shadowed, respectively. The ATG codons at right sides show the initial codons of tdnQ
.
strain UCC22 are carried on the catabolic plasmid
function as genes encoding AD in strain ANA-18.
Frateuria sp. ANA-18 has two catechol-degrading
2
6)
pTDN1, and organize a huge operon with genes
encoding the meta-cleavage pathway (accession no.
D85415). On the other hand, the tdn gene cluster of
1
5)
gene clusters, the cat
ANA-18 tdn gene cluster was found upstream not of
a catabolic plasmid-born cat gene cluster but of the
cat gene cluster, and located on chromosomal DNA.
The cat gene cluster was expressed at a lower concen-
tration of an inducer, cis cis-muconate, than the cat
gene cluster. The location of the tdn and cat gene
clusters and characters of the cat gene cluster may
suggest that aniline is originally metabolized by the
tdn and cat gene clusters, and the cat gene cluster
1
and cat
2
gene clusters. The
strain ANA-18 was independent of cat
1
and cat
2
gene
2
clusters involved in the ortho-cleavage pathway.
Furthermore, a tnpA gene, which encodes trans-
posase and is located upstream of the UCC22 tdn
1
2
,
1
6
)
16)
gene cluster, didn't exist in the same region from
strain ANA-18; ORF7, on the contrary, was located
downstream of tdnR in strain ANA-18. These diŠer-
ences show that the aniline-degrading operons via the
ortho- and meta-cleavage pathways fundamentally
diŠer in their organization in evolutionary processes
although AD genes are derived from a common
ancestor.
1
2
1
2
was acquired as an advantage for rapid catechol
metabolism.
It is reported that the LysR-type regulator BenM
in Acinetobacter sp. ADP1 responds to cis,cis-
The tdn genes were expressed in E. coli and AD
activity was examined. Only E. coli cells carrying
both p23FQ with tdnQ cloned in the low copy vector
pMW218 and p29C11 with tdnTA1A2B showed AD
activity (Table 2). This result shows that the expres-
sion of AD activity needs all components encoded by
tdnQTA1A2B. First, we tried to construct a gene
cluster containing tdnQTA1A2B genes, transcribed
as a single operon by the lac promoter of vector
DNA, but were unable to obtain a recombinant
molecule containing them as the single operon in
spite of several trials. We also failed in the construc-
tion of plasmid DNA containing tdnQTA1 genes,
transcribed by the lac promoter of the vector. Excess
expression of the tdnQ genes may be toxic for E. coli
host cells.
muconate, in addition to benzoate, and activates the
expression of the ben gene cluster, which encodes
enzymes catalyzing the conversion of benzoate to
27)
catechol. However, the production of AD was not
induced in strain ANA-18 by cis cis-muconate
(Fig. 4). This suggests that the LysR-type regulators
TdnR and BenM diŠer in responses to cis cis
,
,
-
muconate although the tdn and ben gene clusters are
involved in the hydroxylation of benzene rings.
Further works on regulation systems of the expres-
sion of the tdn genes from strain ANA-18 are needed
to understand the rapid aniline degradation in this
bacterium.
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