Characterization of a transcriptional regulatory gene involved in dibenzofuran degradation by nocardioides sp. strain DF412

Article abstract of DOI:10.1271/bbb.80496

Nocardioides sp. DF412 degrades dibenzofuran (DF) to salicylate through the sequential actions of DF dioxygenase (dfdA), extradiol dioxygenase (dfdB), and hydrolase (dfdC). The involvement of a TetR-type regulator gene dfdS in the dfdB and dfdS gene expre

Full text of DOI:10.1271/bbb.80496

Biosci. Biotechnol. Biochem., 73 (3), 508–516, 2009
Characterization of a Transcriptional Regulatory Gene Involved
in Dibenzofuran Degradation by Nocardioides sp. Strain DF412
1
1
1
2
1
1;y
Parichat SUKDA, Nao GOUDA, Emi ITO, Keisuke MIYAUCHI, Eiji MASAI, and Masao FUKUDA
1
Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
Department of Civil and Environmental Engineering, Tohoku Gakuin University, Tagajo, Miyagi 985-8537, Japan
2
Received July 18, 2008; Accepted October 31, 2008; Online Publication, March 7, 2009
[doi:10.1271/bbb.80496]
Nocardioides sp. DF412 degrades dibenzofuran (DF)
ing action of a HPHDA hydrolase (Fig. 1A). The
clusters of genes coding angular dioxygenases have
to salicylate through the sequential actions of DF
dioxygenase (dfdA), extradiol dioxygenase (dfdB), and
hydrolase (dfdC). The involvement of a TetR-type
regulator gene dfdS in the dfdB and dfdS gene expres-
sion was investigated. A reporter assay using a lucifer-
ase gene indicated repression of dfdB and dfdS promoter
activities in the presence of the dfdS gene product, DfdS.
Gel shift analysis indicated specific binding of DfdS
to the dfdB promoter region. Both the presence of a
1
0)
been cloned and characterized. Many angular dioxy-
genases are composed of a terminal oxygenase compo-
nent containing large and small subunits and electron
transfer components, including ferredoxin and ferre-
doxin reductase components. The terminal oxygenase
component of carbazole 1,9a-dioxygenase is composed
1
1)
of a homo-trimer of a single subunit. All the genes
encoding the oxygenase and electron transfer compo-
nents are organized in an operonic structure in strain
0
DF-degradation intermediate, 2,2 ,3-trihydroxybiphenyl
0
12)
(
(
2,2 ,3-THBP) or its analog, 2,3-dihydroxybiphenyl
2,3-DHBP), and mutation in the inverted repeat (IR)
YK3.
On the other hand, the terminal oxygenase
component genes are separated from the electron
transfer component genes in strains RW1 and
in the dfdB promoter, canceled repression by DfdS
in vivo and the binding of DfdS to the dfdB promoter
fragment in vitro. These results suggest derepression of
1
3,14)
0
DBF63.
genase and a meta-cleavage compound hydrolase have
Genes encoding a 2,2 ,3-THBP 1,2-dioxy-
0
DfdS in the presence of 2,2 ,3-THBP and 2,3-DHBP and
the involvement of the IR in the repression by DfdS.
been also cloned and characterized, and they are usually
,15–17)
3
located in a single gene cluster.
But the regulatory
system of DF degradation genes has not been reported.
Hence this study was conducted to illustrate the
regulatory mechanism of DF degradation genes in a
DF degrading actinomycete, Nocardioides sp. strain
DF412.
Key words: dibenzofuran; Nocardioides; TetR-type reg-
ulator
Dioxins contain polychlorinated derivatives of diben-
zofuran (DF), dibenzo-p-dioxins (DD), and biphenyl
In strain DF412, the angular dioxygenase genes,
dfdA1A2A3A4, encode large and small subunits of a
terminal oxygenase component, ferredoxin, and ferre-
doxin reductase respectively, and are almost identical to
(
BP), and persist long in the environment. They are
known to be toxic and carcinogenic compounds, and
constitute a group of the most problematic environmental
pollutants. DF has been used as a model compound in the
biodegradation of dioxins. Microorganisms that can
grow on DF have been isolated, including Nocardioides
0
those of strain YK3. The 2,2 ,3-THBP 1,2-dioxygenase
gene, dfdB, and the HPHDA hydrolase gene, dfdC, are
clustered and are located separately from the
18)
1
)
2)
aromaticivorans, Paenibacillus sp. strain YK5,
3
dfdA1A2A3A4 genes. The deduced amino acid se-
)
Rhodococcus sp. strain YK2, Sphingomonas sp. strain
5)
quence of dfdB showed 75% and 74% identity to those
of strains DBF63 and YK2 respectively. That of dfdC
showed 78% and 70% identity to those of strains DBF63
and YK2 respectively. These dfd genes in DF412 are
induced in the presence of DF. An open reading frame
(ORF) located adjacent to and upstream from the dfdB
gene was designated dfdS; it showed similarity to TetR-
type regulatory genes (Fig. 1B). The deduced amino
acid sequence of dfdS showed 18%, 19%, 21%, and 22%
identity to QacR (Swiss-Prot/TrEMBL entry P23217),
TetR (P09164), AcrR (P34000), and CprB (O66122)
respectively. It showed 26% identity to a repressor-like
protein encoded by ORFL6 (Q8VV87), located near
dbfBC in DBF63. The identity of DfdS to other
characterized TetR-type regulators are as low as about
20%, and DfdS appears to be unique among the
4)
HH69, Sphingomonas wittichii RW1, and Terrabacter
6
)
7)
3)
sp. strains DBF63, DPO360, and YK3. Some of
these have been found to co-metabolize chlorinated
8
,9)
derivatives of DF and DD.
In aerobic DF degradation, metabolism of DF is
initiated by hydroxylation at 4,4a position, which is
catalyzed by a ring-hydroxylating dioxygenase. This
type of reaction is classified as angular dioxygenation.
After hydroxylation, the product is spontaneously trans-
0
0
formed to 2,2 ,3-trihydroxybiphenyl (2,2 ,3-THBP). The
0
aromatic ring of 2,2 ,3-THBP is cleaved by a meta-
0
cleavage dioxygenase, 2,2 ,3-THBP 1,2-dioxygenase,
0
and the resulting 2-hydroxy-6-(2 -hydroxyphenyl)-6-
oxohexa-2,4-dienoate (HPHDA) is converted to salicy-
late and 2-hydroxypenta-2,4-dienoic acid by the follow-
y
To whom correspondence should be addressed. Fax: +81-258-47-9450; E-mail: masao@vos.nagaokaut.ac.jp
0
0
Abbreviations: DF, dibenzofuran; 2,3-DHBP, 2,3-dihydroxybiphenyl; 2,2 ,3-THBP, 2,2 ,3-trihydroxybiphenyl

TetR-Type Regulator for Dibenzofuran Degradation
509
DNA manipulations and analysis. DNA manipulations were
22)
A
performed essentially as described by Ausubel et al. and Sambrook
2
3)
et al. To transform R. erythropolis IAM1399, electroporation was
used.24) Nucleotide sequences were determined by the dideoxy
termination method with a CEQ 2000XL genetic analysis system
(
Beckman Coulter, Fullerton, CA). The Sanger reaction25) was carried
out using a CEQ dye terminator cycle sequencing quick start kit
Beckman Coulter). Analysis of the nucleotide sequence was
(
performed with the GeneWorks program (Intelligenetics, Mountain
View, CA).
Plasmid construction. Plasmids used in this study are listed and
described in Table 1. Deletion derivatives of the dfdB upstream region
0
were constructed by PCR using primer PdfdBF51 (5 -AGAA-
0
0
CTAGTTTGAATGGAACGTTCGCTTC-3 ) or PdfdBF76 (5 -AG-
0
AACTAGTCCGCGCTATGTGACCCTCGT-3 ) in combination with
0
0
pKLASB-R (5 -AGAACTAGTCTGCAGGCATGCAAGCTGGT-3 ),
0
0
or primer PdfdBF88 (5 -GTCGACTTGACGGGAGCGCCGC-3 ) or
0
0
PdfdBF188 (5 -GTCGACCGACCTCGCCGATTCCC-3 ) in combina-
0
0
tion with PdfdBS (5 -GTCGACCCGCTCATGTGATGTCC-3 ), as
0
indicated in Table 1. Primers IR dfdS-F (5 -ATTGAATGGAACG-
0
0
TTCGCGGTAAAACTGGACGGAAGGACA-3 ) and IR dfdS-R (5 -
0
TGTCCTTCCGTCCAGTTTTACCGCGAACGTTCCATTCAAT-3 )
were used to perform site-directed mutagenesis of IR.
B
Assay for DfdB activity. R. erythropolis IAM1399 transformants
were grown in 0.2 ꢁ LB containing appropriate antibiotics, and then
were washed with 0.2 ꢁ LB medium and suspended in 0.2 ꢁ LB
medium containing appropriate antibiotics at A600 of 1.0. Ten
ꢀ
milliliters of each cell suspension was incubated at 30 C for 3 h with
appropriate inducers. Cells were collected and disrupted by sonication,
and the cell debris was removed by centrifugation. The supernatant
(
cell extract) was used in enzymatic assays. The 2,3-DHBP dioxyge-
Fig. 1. Proposed Degradation Pathway of DF (A), and Genetic
Organization of the DF Degradation Genes in Nocardioides sp.
DF412 (B).
The genes responsible for each enzyme step are indicated, and
directions of transcription are indicated by arrows. The proposed
function of each gene product is described below the gene names.
nase activities were assayed in 1 ml of cell suspension containing 10 mg
of protein and 2 mM 2,3-DHBP at 30 C. Absorption at 434 nm, derived
ꢀ
from the metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid,
was monitored with a Beckman DU-7500 spectrophotometer (Beck-
man Coulter).
Luciferase assay. R. erythropolis IAM1399 transformants grown on
.2 ꢁ LB medium containing appropriate antibiotics were washed
0
characterized members of the TetR family. The amino
acid residues from 16 to 58 of DfdS exhibits high
identity of 35% with CprB. The corresponding residues
of CprB were estimated to constitute ꢀ helices 1 to 3
based on the three-dimensional structure, and the
residues from helices 2 to 3 comprise the DNA-binding
helix-turn-helix.1 The corresponding residues from 41
to 48 of DfdS are predicted to form the helix-turn-helix,
which recognizes the target DNA sequence in a target
promoter region.
with 0.2 ꢁ LB medium and suspended in 0.2 ꢁ LB medium contain-
ing appropriate antibiotics at A600 of 1.0. Ten milliliters of each cell
ꢀ
suspension was incubated at 30 C for 3 h in the absence and the
presence of a candidate for the inducer compound. DF and 2,3-
DHBP were supplied at a final concentration of 100 mM unless
otherwise specified. The solution of 2,2 ,3-THBP was prepared using
0
R. erythropolis IAM1399 harboring pK4PA2A1234 and pFST1. This
strain expresses DF dioxygenase, DfdA1A2A3A4, in the presence of
ethylbenzene. The resting cells of this strain were incubated with 50 mM
of DF, and after the disappearance of DF was confirmed, the
9)
0
supernatant was used as 50 mM 2,2 ,3-THBP solution. R. erythropolis
IAM1399, harboring pK4PA2 and pFST1, was used in preparing a
In this study, we investigated the involvement of dfdS
in the regulation of dfd gene expression.
negative control solution. After incubation with substrates, the cell
suspension was diluted to 1:10 in lux buffer.32) Twenty microliters of
lux buffer containing 0.1% (vol/vol) 1-decanal was added to 80 ml of
diluted cell suspension, and the luminescence for 5 s was measured
using a Wallac 1420 Multilabel Counter (PerkinElmer, Waltham, MA).
The turbidity at A600 of the cell suspension was also measured, and the
relative luminescent unit (RLU) was calculated by dividing the
luciferase activity by the turbidity.
Materials and Methods
Bacterial strains, plasmids, and culture conditions. Bacterial strains
and plasmids used in this study are listed in Table 1. Nocardioides sp.
ꢀ
DF412 was grown at 30 C in Luria-Bertani (LB) medium (10 g of
Bacto Tryptone (Difco Laboratories, Detroit, MI) per liter, 5 g of yeast
extract (Difco Laboratories) per liter, 5 g of NaCl per liter), 0.2 ꢁ LB
medium (2 g of Bacto Tryptone per liter, 1 g of yeast extract per
liter, 5 g of NaCl per liter) or W minimal medium20) containing
Primer extension analysis. The transcriptional start sites of dfdS and
dfdB were determined by DNA fragment analysis using a CEQ2000
0
system (Beckman Coulter). Primers PEXdfdS (5 -GCGCATGGC-
0
0
50 mM dibenzofuran (DF) as the sole carbon source. Rhodococcus
erythropolis IAM1399 (ATCC 15963) was grown at 30 C in LB or
GGCGAAGTAGTCCC-3 ) and PEXdfdB (5 -GCTCTCGACCTCG-
0
ꢀ
TAGCCGCTGG-3 ), with specific WellRED dye (Sigma-Aldrich,
0
.2 ꢁ LB medium. DF and 2,3-dihydroxybiphenyl (2,3-DHBP) were
St. Louis, MO), were used in the extension. Total RNAs from
Nocardioides sp. DF412 and R. erythropolis IAM1399 were prepared
using Isogen (Nippon Gene, Toyama, Japan). After removal of DNA
contamination with RNase-free DNase I (Takara Bio, Otsu, Japan),
RNAs were recovered by ethanol precipitation. One hundred pmol of
the primer was incubated with 20 mg of RNAs in hybridization buffer
dissolved in dimethyl sulfoxide (DMSO), and were added to the media.
Escherichia coli JM10921) was grown at 37 C in LB medium. In
cultures of cells carrying antibiotic resistance markers, the medium
was supplemented with 100 mg of ampicillin/liter, 50 mg of kanamy-
cin/liter, or 20 mg of chloramphenicol/liter.
ꢀ

510
P. SUKDA et al.
Table 1. Strains and Plasmids Used in This Study
Strain or plasmid
Relevant characteristicsa
Reference or origin
Strains
þb
ꢂb
Nocardioides sp. DF412
Rhodococcus erythropolis
IAM1399 (ATCC 15963)
E. coli JM 109
Wild type; DF
Wild type; DF
This study
IAM culture collectionc
0
recA1 supE44 endA1 hsdR17 gyrA96 relA1 thiꢀ (lac-proAB)/F
traD36 proAB lacIq lacZꢀM15]
þ
21
[
Plasmids
þ
A cloning vector, Apr
pYN17 with 900-bp BamHI fragment carrying dfdS
pBluescript II KS
pBPdfdS
pBSL
Stratagene
This study
26
þ
pBluescript II KS with new SpeI site in place of KpnI site
r
pFAJ2574
pFPdfdS
pFST1
A Rhodococcus-E. coli shuttle vector, Cm
27
This study
28
pFAJ2574 with SpeI fragment of pBPdfdS carrying PaphII-dfdS
pFAJ2574 with 6.2-kb fragment carrying bphST
A cloning vector, Apr
pGEM-T
pGEMBS167
Promega
This study
pGEMBS188 containing 167-bp promoter region of dfdB obtained
by the deletion of 21-bp SmaI fragment
pGEMBS188
pGEM-T containing188-bp promoter region of dfdS and dfdB amplified
with the primers PdfdBF188 and PdfdBS
This study
pKdfdS-R
pKdfdS-RꢀS
pKLA1
pK4 with 2.5-kb PstI fragment carrying dfdS and dfdB
pKdfdS-R with a deletion of SgrAI segment in dfdS
A promoter probe vector containing luciferase structural genes, luxAB
from V. harveyi, Kmr
This study
This study
29
pKLASB1
pKLASB2
pKLASB3
pKLASB5
pKLA1 with the insertion of 188-bp promoter region of dfdB
from pGEMBS188 at SalI site
This study
This study
This study
This study
pKLA1 with the insertion of 188-bp promoter region of dfdS
from pGEMBS188 at SalI site
pKLA1 containing 167-bp deleted promoter region of dfdB
from pGEMBS167
pKLA1 with the insertion of 88-bp promoter region of dfdB
from pT7-88 at SalI site
pKLASB5mut
pKLASB7
pKLA1 containing 88-bp mutant promoter region of dfdB
pKLA1 containing 76-bp promoter region of dfdB amplified
with the primers PdfdBF76 and pKLASB-R
pKLA1 containing 51-bp promoter region of dfdB amplified
with the primers PdfdBF51 and pKLASB-R
A Rhodococcus-E. coli shuttle vector, Kmr
This study
This study
pKLASB9
This study
pK4
30
31
pK4PA2
pK4PA2A1234
pT7Blue T
pT7-88
pK4 carrying the promoter region of bphA1
pK4PA2 carrying dfdA1, dfdA2, dfdA3 and dfdA4 genes
A cloning vector, Apr
This study
Novagen
This study
pT7Blue T containing 88-bp promoter region of dfdB amplified
with the primers PdfdBF88 and PdfdBS
A mutant derivative of pT7-88
pT7-88mut
pYN17
pYN24
This study
This study
This study
pBSL carrying promoter region of aphII gene (PaphII)
pFAJ2574 with SpeI fragment containing PaphII from pYN17
a
r
r
r
Abbreviations: Km , Cm , and Ap , resistance to kanamycin, chloramphenicol, and ampicillin respectively
bDF, growth on dibenzofuran
cIAM, Institute of Applied Microbiology
ꢀ
(
1 mM Tris–HCl, pH 8.3, 0.1 mM EDTA, 0.015 M KCl) at 80 C for
pH 7.6, 5 mM EDTA, 50 mM (NH4)2SO4, 5 mM DTT, 1% (w/v) Tween
20, 150 mM KCl), 1 ml of poly(dI-dC), and 1 ml of poly-(L)-lysine; total
assay volume, 10 ml. When needed, DF or 2,3-DHBP in DMSO was
added to the reaction mixture. Then the mixtures were subjected to
electrophoresis on a 6% polyacrylamide gel with 0.5 ꢁ TBE running
buffer. The DIG-oligonucleotide/protein complexes were transferred
to a Hybond-N blotting membrane (Amersham Biosciences, Buck-
inghamshire, UK), and the shifted bands were visualized according to
the attached protocol.
15 min, and was allowed to cool. After recovery of RNA hybridized
with the primer by ethanol precipitation, reverse-transcription was
ꢂ
performed with M-MLV Reverse Transcriptase RNaseH (Toyobo,
ꢀ
Osaka, Japan). RT reactions were performed at 30 C for 11 min and at
ꢀ ꢀ
4
2 C for 45 min, followed by heating at 99 C for 5 min. The
synthesized DNA was recovered by ethanol precipitation, and was
dissolved in 25 ml of SLS buffer containing 0.125 ml of the CEQ DNA
Size Standard-400 (Beckman Coulter). Samples were analyzed by
CEQ2000XL with fragment analysis software (Beckman Coulter).
Detection of gene product. Expression of dfdS and dfdB in
R. erythropolis IAM1399 transformants was examined by sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, as described
previously.33) Transformants were incubated in 10 ml of LB containing
Gel shift analysis. Plasmid pGEMBS188 was digested by SalI to
recover a 188-bp promoter fragment that was identical to that in
pKLASB1. The 88-bp and mutated 88-bp fragments, identical to those
in pKLASB5 and pKLASB5mut respectively, were obtained using
ꢀ
1% glycine and appropriate antibiotics at 30 C until the optical density
0
primers PdfdBF88 and PdfdBS. The fragments were 3 -end-labelled
with digoxigenin-11-ddUTP by terminal transferase using a DIG gel
shift kit (Roche Diagnostics, Basel, Switzerland). The labeled probes
of the cultures reached an A600 of 1.0. The addition of glycine at high
concentration is known to accelerate efficient cell lysis in Actino-
bacteria.34) The cells were then washed with 50 mM sodium phosphate
buffer (pH 7.0), and re-suspended in the same buffer. After disruption
by sonication and removal of cell debris by centrifugation, the protein
concentration was determined with a protein assay kit (Bio-Rad
(
48 fmol of double-stranded oligonucleotides) were incubated for
ꢀ
2
0 min at 20 C with various concentrations of crude cell extract in a
mixture including 2 ml of 5ꢁ binding buffer (100 mM HEPES buffer,

TetR-Type Regulator for Dibenzofuran Degradation
511
A
A
C
B
B
C
Fig. 3. Complementation of dfdS Deletion.
A, Physical map of pFPdfdS. B, DfdB activity of R. erythropolis
IAM1399 harboring pKdfdS-RꢀS plus pFPdfdS or pYN24. C,
SDS–PAGE analysis of IAM1399 transformants. Each cell extract
(
10 mg) was separated in an SDS-12% polyacrylamide gel and
stained with Coomassie Brilliant Blue. Plasmids introduced into
IAM1399 are indicated above each lane. Lane M, molecular size
markers. The protein band corresponding to DfdB is indicated by an
arrowhead.
Fig. 2. Influence of dfdS on dfdB Expression.
A, Physical maps of pKdfdS-R and pKdfdS-RꢀS. The SgrAI-
SgrAI segment in pKdfdS-R is deleted in pKdfdS-RꢀS. B, DfdB
activity of R. erythropolis IAM1399 harboring pKdfdS-R or
pKdfdS-RꢀS. The substrate was added at 0 s. C, SDS–PAGE
analysis of the IAM1399 harboring pKdfdS-R or pKdfdS-RꢀS.
Each cell extract (10 mg) was separated in an SDS-12% polyacryl-
amide gel and stained with Coomassie Brilliant Blue. Lane 1,
molecular size markers; lanes 2 and 3, crude extract of IAM1399
harboring pKdfdS-R and pKdfdS-RꢀS respectively. The protein
band corresponding to DfdB is indicated by an arrowhead.
formed to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid (HPDA) with absorption at 434 nm. The DfdB
activity of the cell extract of R. erythropolis IAM1399
harboring pKdfdS-RꢀS (17.8 U/mg) was approximately
50-fold higher than that harboring pKdfdS-R (0.36
U/mg) (Fig. 2B). Strong expression of the DfdB protein
in IAM1399 harboring pKdfdS-RꢀS was observed on
sodium dodecyl sulfate-polyacrylamide gel electropho-
resis (SDS–PAGE) analysis (Fig. 2C). The protein band
corresponding to DfdB was not observed in IAM1399
harboring pKdfdS-R. These results suggest repression of
DfdB expression by DfdS. To confirm the involvement
of dfdS in the repression of dfdB expression, the deleted
dfdS gene in pKdfdS-RꢀS was trans-complemented
with a plasmid pFPdfdS, which contains intact dfdS
under the control of the aphII gene promoter (Fig. 3A).
In the transformant harboring this plasmid, DfdS was
considered to be constitutively expressed. Both the
DfdB activity and the production of the DfdB protein
were repressed in a trans-complemented strain harboring
both pKdfdS-RꢀS and pFPdfdS (Fig. 3B and C). On
the other hand, no repression occurred in the strain
harboring pKdfdS-RꢀS and a vector plasmid, pYN24.
To determine the transcriptional repression of dfdB, we
employed a promoter probe vector, pKLA1, carrying
promoter-less luxAB luciferase genes. The 188-bp
promoter region of dfdB was inserted upstream from
the adjacent luxAB genes in pKLA1 to construct a
reporter plasmid, pKLASB1 (Fig. 4A). When R. eryth-
ropolis IAM1399 was co-transformed with pKLASB1
and pFPdfdS, DfdS-dependent repression of luciferase
Laboratories, Richmond, CA) by the method of Bradford,35) and 10 mg
of protein was electrophoresed.
Site-directed mutagenesis. Site-directed mutagenesis was performed
ꢀ
with the QuickChange Site-Directed Mutagenesis kit (Stratagene, La
Jolla, CA). Primers IR dfdS-F and IR dfdS-R were used, and plasmid
pT7-88 was used as the template. The resulting mutated fragment was
inserted into pKLA1 to construct pKLASB5mut.
Results and Discussion
Influence of dfdS on expression of the dfdB gene
The dfdS gene was 651-bp long, and the deduced
molecular weight of its product was 23,290. To inves-
tigate the involvement of dfdS in dfdB transcription,
pKdfdS-R with the native dfdS and dfdB loci and
pKdfdS-RꢀS with a deletion in the dfdS gene were
constructed (Fig. 2A). Each plasmid was introduced into
a host strain, R. erythropolis IAM1399, which belongs
to the same actinomycetales as Nocardioides. The
expression of dfdB in the transformants was examined
by measuring DfdB activity toward 2,3-dihydroxybi-
phenyl (2,3-DHBP), which was expected to be trans-

512
P. SUKDA et al.
A
B
C
D
E
Fig. 4. Promoter Analysis of dfdB and dfdS.
A, DNA inserts of pKLASB1, pKLASB2, pKLASB3, pKLASB5, pKLASB7, and pKLASB9. The various inserts were connected to luxAB of
pKLA1 as illustrated. B, Substrate preference of DfdS. The reporter plasmid used was pKLASB1. C, Luciferase activities of R. erythropolis
IAM1399 harboring a reporter plasmid plus pFPdfdS or pYN24 in the absence and the presence of 2,3-DHBP. D, Effects of mutation on the IR
sequence of the dfdB promoter. The reporter plasmids used are indicated below. E, Luciferase activities of R. erythropolis IAM1399 harboring
a
0
pKLASB2 plus pFPdfdS or pYN24 in the absence and the presence of substrates. 2,2 ,3-THBP was supplied as supernatant of the
R. erythropolis IAM1399 cell culture harboring pK4PA2A1A2A3A4 and pFST1, incubated with DF. The graphs denote the averages of
triplicate experiments with standard error bars.
0
activity was observed (Fig. 4B). These results indicate
that DfdS represses the transcription of the dfdB gene,
and that the 188-bp fragment between dfdB and dfdS
contains a DfdS responsive element and a dfdB promoter
sequence. This 188-bp sequence had no outstanding
similarity to the sequences upstream from the meta-
cleavage enzyme genes of DBF63, DPO360, and YK2.
Using this luxAB reporter system, the effector substrate
preference of DfdS was investigated with DF, 2,3-DHBP
by 2,3-DHBP and 2,2 ,3-THBP, suggesting that the
0
effector for DfdS is 2,2 ,3-THBP, a metabolite of DF
(Fig. 4B). It is unclear why the addition of 2,3-DHBP
and of 2,2 ,3-THBP did not restore the luciferase activity
of IAM1399 expressing DfdS as did that of IAM1399
harboring the vector control. High dosage expression of
DfdS by the multicopy vector, or inefficient uptake of
2,3-DHBP and 2,2 ,3-THBP into the cytoplasm might
have caused these results.
0
0
0
0
0
and 2,2 ,3-trihydroxybiphenyl (2,2 ,3-THBP). 2,2 ,3-
THBP is a metabolic intermediate of DF. It was prepared
from DF using R. erythropolis IAM1399 expressing
DfdA1A2A3A4. 2,3-DHBP is a metabolic intermediate
Promoter analysis of dfdB
To address the promoter sequence of dfdB, promoter
deletion analysis was performed. The various deleted
promoter fragments were inserted into pKLA1
(Fig. 4A), and repression by DfdS was examined by
measuring the luciferase activities of R. erythropolis
IAM1399 harboring the various deletion plasmids and
pFPdfdS. The results indicated that DfdS repressed the
0
of biphenyl, and it has a structure analogous to 2,2 ,3-
THBP. Each of these substrates was added to the cell
culture of IAM1399 harboring pKLASB1 and pFPdfdS,
and luciferase activities were measured after incubation
ꢀ
for 3 h at 30 C (Fig. 4B). Repression by DfdS was eased

TetR-Type Regulator for Dibenzofuran Degradation
513
Fig. 5. Nucleotide Sequences of the Promoter Regions of dfdS and dfdB.
The sequence of the dfdS-dfdB intergenic region is shown. The primer sequences used in the primer extension experiment are underlined. The
open bent arrows represent the borders of subcloned fragments in the deletion derivatives presented in Fig. 4A. The transcriptional start sites are
indicated by closed bent arrows and +1, and the inverted repeat is indicated by arrows. The translational start codons for dfdB and dfdS are
represented in bold. The open box indicates the position of site-directed mutagenesis, and the mutated sequence is indicated above the box.
luciferase activities of R. erythropolis IAM1399 harbor-
ing pKLASB3, pKLASB5, or pKLASB7 as well as that
harboring pKLASB1, suggesting that the 76-bp frag-
ment inserted in pKLASB7 is sufficient for repression by
DfdS (Fig. 4C). This repression was eased in the
presence of 2,3-DHBP among IAM1399 derivatives
harboring pKLASB3 or pKLASB5 as well as that
harboring pKLASB1. Derepression was not strong, and
the luciferase activity of IAM1399 harboring pKLASB5
and pFPdfdS was elevated 3-fold in the presence of
which was not effective with RNA from DF412. This
peak did not appear when using cDNA derived from
IAM1399 carrying pKdfdS-R, which contains intact
dfdS. The upstream sequence from the transcriptional
start site of the dfdB gene did not show similarities to the
ꢂ35 and ꢂ10 sequences of the E. coli promoter. TetR-
type regulators have been reported to bind to inverted
repeats (IRs) in the operator region. In the 88-bp
fragment, there was an IR which partially overlapped the
transcriptional start site of the dfdB gene (Fig. 5),
suggesting that the binding of DfdS to this IR results in
repression of transcription. To confirm the binding of
DfdS to the IR, site-directed mutagenesis was per-
formed. The 3-bp nucleotides located in the IR were
changed from TTC to GGT to construct pKLASB5mut
(Fig. 5). Repression was not observed in R. erythropolis
IAM1399 harboring pKLASB5mut and pFPdfdS, sug-
gesting that the IR is the binding site of DfdS (Fig. 4D).
3
7)
2
,3-DHBP. Thus the dfdB promoter activities of the
derepressed cells harboring pKLASB1, pKLASB3, or
pKLASB5 were far less than that of the cells without the
dfdS gene. Such incomplete derepression in vivo was
also observed with ttgR, a tetR-type repressor control-
ling the antibiotic efflux system encoded by ttgABC in
3
6)
Pseudomonas putida DOT-T1E. No derepression was
observed in the cells harboring pKLASB7 and pFPdfdS,
suggesting that the 88-bp fragment inserted in
pKLASB5 is required for derepression by 2,3-DHBP.
The luciferase activity of IAM1399 harboring the
various reporter plasmids individually plus vector
pYN24 in the presence of 2,3-DHBP was almost the
same as that in the absence of 2,3-DHBP (data not
shown). The luciferase activity of IAM1399 harboring
pKLASB9 with the 51-bp insertion was always low,
suggesting damage in the core promoter sequence
Influence of DfdS on the dfdS promoter
To investigate the transcription of dfdS, we performed
promoter analysis of dfdS. We constructed pKLASB2
carrying the same fragment as pKLASB1 in the opposite
direction (Fig. 4A), and the luciferase activities of
R. erythropolis IAM1399 harboring pKLASB2 and
pFPdfdS or pYN24 were measured. The basal promoter
activity of dfdS was lower than that of dfdB, and
transcription from the dfdS promoter was repressed to
approximately one third in the presence of DfdS
(Fig. 4E), suggesting autogenous repression by DfdS.
The promoter activity of dfdS in the presence of
2,3-DHBP was mostly the same as in the absence of
2,3-DHBP, but derepression in the presence of 2,3-
DHBP was estimated to be incomplete and weak as
indicated for the dfdB promoter, and repression of the
dfdS promoter by DfdS itself was small. Thus derepres-
sion of the dfdS promoter can be too small to detect.
To identify the transcriptional start site of dfdS, we
performed primer extension analysis with total RNAs
extracted from DF412. cDNA synthesis was carried out
with the PEXdfdS primer (Fig. 5). The nucleotide
located 330-bp from the primer was indicated to be
(
Fig. 4C).
The transcriptional start site of dfdB was determined
by primer extension analysis. Initially, total RNA
prepared from DF412 grown on DF and the primer
PEXdfdB was used, but no specific peak for the
transcriptional start site of dfdB was obtained. Then
we employed total RNA from IAM1399 carrying
pKdfdS-RꢀS. cDNA synthesis was carried out with
the same primer, and the product gave a single peak that
mapped 245-bp away from the primer (data not shown),
indicating that the transcriptional start site of dfdB is
located 35 bp upstream from the putative start codon of
dfdB. The dfdB segment in the high copy number
plasmid might have enabled us to obtain the peak for the
transcriptional start site of dfdB using primer PEXdfdB,

514
P. SUKDA et al.
the major transcriptional start site (data not shown),
which was 84-bp upstream from the putative dfdS start
codon (Fig. 5).
A
Gel shift analysis
The in vitro interaction between the dfdB promoter
region and DfdS was investigated by gel shift assay. The
0
1
88-bp 3 -end-labelled DNA fragment, which was the
identical fragment carried by pKLASB1, was used as
a probe. DfdS was supplied as a cell-free extract of
R. erythropolis IAM1399 carrying pFPdfdS. Shifted
bands corresponding to complexes I and II were
observed when DfdS-expressing cell-free extract was
used, and no shifted band was observed when the vector
control was used (Fig. 6A). The intensity of the band
corresponding to complex II increased in a dose-
dependent manner responding to the amount of DfdS-
expressing cell extract. The appearance of complex I
appeared to be independent of the dosage of DfdS.
These results indicate that DfdS binds to the dfdS-dfdB
intergenic region in vitro. We also observed that
B
2
,3-DHBP but not DF released the binding of DfdS,
suggesting that 2,3-DHBP is an inducer for the der-
epression of DfdS (Fig. 6B), which confirms the results
in vivo for IAM1399 harboring pKLASB1 and pFPdfdS
(
Fig. 4B). To determine whether repression of dfdB by
C
DfdS occurs via direct physical interaction between the
dfdB promoter region and DfdS, gel shift analysis was
performed on a 3 -end-labelled mutated 88-bp DNA
0
fragment containing the intergenic dfdB-dfdS region
amplified from pT7-88mut, which was identical to the
inserted fragment in pKLARB5mut. Shifted bands were
observed when the DfdS-expressing cell-free extract and
wild-type 88-bp was used (Fig. 6C, lane 6), and the
binding of DfdS was released in the presence of 2,3-
DHBP (Fig. 6C, lane 7). In the mutant 88-bp fragment,
the shifted band was not observed even in the presence
of DfdS (Fig. 6C, lane 3). These results indicate that the
IR sequence is involved in the repression of the dfdB
promoter by DfdS. The DfdS binding sequence was
found to be a 22-bp IR with a pair of 11-bp half sites. It
was estimated that TetR binds to a 15-bp IR as a dimer,
and that each TetR molecule specifically recognizes a
Fig. 6. Gel Shift Analysis of the dfdB Promoter.
A, Binding of DfdS to the 188-bp dfdB promoter fragment. The
probe was incubated with cell extract of R. erythropolis IAM1399
harboring pYN24 (lanes 2–5) or pFPdfdS (lanes 6–9). The amounts
of proteins were 0.5 mg (lanes 2 and 6), 1 mg (lanes 3 and 7), 5 mg
(
lanes 4 and 8), and 12 mg (lanes 5 and 9). Lane 1, 188-bp free probe.
B, The effect of DF and 2,3-DHBP on the binding of DfdS. The 188-
bp probe and 1 mg of the cell extract of R. erythropolis IAM1399
harboring pFPdfdS were incubated with nothing (lane 2), DMSO
(lane 3), 2,3-DHBP (lanes 4, 5, and 6), or DF (lanes 7, 8, and 9). The
concentrations of 2,3-DHBP and DF were 10 mM (lanes 4 and 7),
-bp sequence constituting a half site of IR.38) On the
6
other hand, QacR was found to bind to a 36-bp target
sequence with a pair of 12-bp half sites of IR separated
by 6-bp spacer as a couple of dimers, and each dimer
was estimated specifically to recognize a 10-bp sequence
1
00 mM (lanes 5 and 8), and 1 mM (lanes 6 and 9). Lane 1, 188-bp
free probe. C, Binding of DfdS to the dfdB promoter fragment and
its mutant. The wild-type probes (lanes 5–7) and the mutant probes
(lanes 2 and3) were incubated with the cell extract of R. erythropolis
IAM1399 harboring pYN24 (lanes 2 and 5) or pFPdfdS (lanes 3 and
3
9)
constituting a half site of IR. The 22-bp IR suggests
that DfdS binds to the IR as a couple of dimers. This
IR overlaps the region from ꢂ4 to ꢂ25 of the dfdS
promoter and that from the transcription start to ꢂ8 of
the dfdB promoter. Similar positioning of IR in the
region including ꢂ10 has been reported in other TetR
family members, such as ttgR40) and qacR. No ap-
parent sequence conservation was observed among
6
). Lane 7, the probe and the cell extract were incubated in the
presence of 10 mM 2,3-DHBP. Lanes 1 and 4, free probes. The
amount of protein in each cell extract was 5 mg.
ing of inducers to them.41) The derepression mechanism
of DfdS, however, appears to be more complicated.
First, derepression of the dfdB promoter was apparently
incomplete, although the results of gel shift analysis
suggested complete release of DfdS from the IR in the
presence of an inducer. Similar contradictory results
37)
4
0)
37)
the binding sequences of DfdS, TtgR, QacR, and
TetR.3
8)
The results presented here indicate repression of
transcription from the dfdB promoter by DfdS through
binding of DfdS to the IR in dfdB promoter in the
4
0)
have also been reported for TtgR protein.
The
presence of excess repressor and inducer in gel shift
analysis in vitro appears to have brought about results
inconsistent with those in vivo. Second, the border of the
88-bp insert in pKLASB5 spanning from ꢂ33 to ꢂ45
0
absence of an inducer, 2,2 ,3-THBP, as previously found
for TetR and QacR, which exert derepression by
dissociation from their target promoters upon the bind-

TetR-Type Regulator for Dibenzofuran Degradation
515
of the dfdB promoter, which is separated from the IR
by 25-bp, was found to be required for derepression.
This region contains a nucleotide sequence, TTGAC-
GGGAGC, which is similar to the half site of IR,
TTGAAGCGAAC. Interaction of another cellular factor
or DfdS with this region can be involved in the efficient
derepression in vivo.
terminal oxygenase component of angular dioxygenase, carba-
zole 1,9a-dioxygenase. J. Mol. Biol., 351, 355–370 (2005).
2) Iida, T., Mukouzaka, Y., Nakamura, K., and Kudo, T., Plasmid-
borne genes code for an angular dioxygenase involved in
dibenzofuran degradation by Terrabacter sp. strain YK3. Appl.
Environ. Microbiol., 68, 3716–3723 (2002).
1
13) Bunz, P. V., and Cook, A. M., Dibenzofuran 4,4a-dioxygenase
from Sphingomonas sp. strain RW1: angular dioxygenation by a
three-component enzyme system. J. Bacteriol., 175, 6467–6475
(
1993).
Acknowledgments
1
4) Kasuga, K., Habe, H., Chung, J.-S., Yoshida, T., Nojiri, H.,
Yamane, H., and Omori, T., Isolation and characterization of the
genes encoding a novel oxygenase component of angular
dioxygenase from the gram-positive dibenzofuran-degrader
Terrabacter sp. strain DBF63. Biochem. Biophys. Res. Com-
mun., 283, 195–204 (2001).
We thank Prof. Ren e´ De Mot for the kind gift of
plasmid pFAJ2574. This study was supported in part by
the 21st Century COE (Center for Excellence) Program
of Nagaoka University of Technology (NUT), a Grant-
in-aid (Hazardous Chemicals) from the Ministry of
Agriculture, Forestry, and Fisheries of Japan (HC-05-
1
5) Happe, B., Eltis, L. D., Poth, H., Hedderich, R., and Timmis,
0
K. N., Characterization of 2,2 ,3-trihydroxybiphenyl dioxyge-
nase, an extradiol dioxygenase from the dibenzofuran- and
dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain
RW1. J. Bacteriol., 175, 7313–7320 (1993).
2
322-1), and Grants-in-aid for the Encouragement of
Young Scientists (18780052) and for Scientific Research
on Priority Areas ‘‘Comparative Genomics’’ from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan.
16) Kasuga, K., Nojiri, H., Yamane, H., Kodama, T., and Omori, T.,
Cloning and characterization of genes involved in the degrada-
tion of dibenzofuran by Terrabacter sp. the strain DBF63.
J. Ferment. Bioeng., 84, 387–399 (1997).
1
7) Schmid, A., Rothe, B., Altenbuchner, J., Ludwig, W., and
Engesser, K.-H., Characterization of three distinct extradiol
dioxygenases involved in mineralization of dibenzofuran by
Terrabacter sp. strain DPO360. J. Bacteriol., 179, 53–62
(1997).
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