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BphS, a key transcriptional regulator of bph genes involved in
PCB/biphenyl degradation in Pseudomonas sp. KKS102
YOSHIYUKI OHTSUBO*, MINA DELAWARY, KAZUHIDE KIMBARA+,
MASAMICHI TAKAGI, AKINORI OHTA, and YUJI NAGATA#
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan.
+Railway Technical Research Institute, 2-8-38 Hirari-cho, Kokubunji-shi,
Tokyo 185-8540, Japan.
#Institute of Genetic Ecology, Tohoku University, Katahira, Sendai 980-8577,
Japan.
*Present address for corresponding author: Laboratory of Microbiology,
RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako,
Saitama 351-0198, Japan; Phone: +81-48-467-9544; Fax: +81-48-462-4672; E-
mail: [email protected]
Running Title: Regulation of bph genes in Pseudomonas sp. KKS102
Keywords: transcriptional regulation, bph genes, repression, Pseudomonas sp.
KKS102, PCB, biphenyl
Summary
The bph genes in Pseudomonas sp. KKS102, which are involved in the
degradation of PCB/biphenyl, are induced in the presence of biphenyl. In this
study our goal was to understand the regulatory mechanisms involved in the
inducible expression. The bph genes (bphEGF(orf4)A1A2A3BCD(orf1)A4R)
constitutes an operon, and its expression is strongly dependent on the pE
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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promoter located upstream of the bphE gene. A bphS gene, whose deduced
amino acid sequence showed homology with GntR family transcriptional
repressors, was identified at the upstream region of the bphE gene. Disruption
of the bphS gene resulted in constitutive expression of bph genes, suggesting
that the bphS gene product negatively regulated the pE promoter. The gel
retardation and DNase footprinting analyses demonstrated specific binding of
BphS to the pE promoter region and identified four BphS binding sites that
were located within and immediately downstream of the -10 box of the pE
promoter. The four binding sites were functional in repression, because their
respective elimination resulted in derepression of pE promoter. The binding
of BphS was abolished in the presence of 2-hydroxy-6-oxo-6-phenylhexa-2,4-
dienoic acid, an intermediate compound in the biphenyl-degradation pathway.
We concluded that the negative regulator BphS plays a central role in the
regulation of bph gene expression through its action at the pE promoter.
Introduction
Human activities have created toxic compounds that cause environmental
pollution and threaten the earth's biosphere. Among these compounds, PCB is
one of the most serious pollutants, and the microorganisms capable of degrading
PCB have been studied worldwide (1) (2). Pseudomonas sp. KKS102 has been
isolated (3) and shown to degrade PCB/biphenyl via a meta-cleavage pathway to
yield a tricarboxylic acid cycle intermediate and benzoic acid (4) (5) (6) (7)
(Fig. 1A). The genes coding enzymes for this conversion have been sequenced,
characterized, and shown to be clustered as
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bphEGF(orf4)A1A2A3BCD(orf1)A4R (Fig. 1B).
In many microorganisms capable of degrading chemical compounds, the
transcription of genes involved in the degradation is regulated (8). In most
cases, the genes coding for the regulator exist near the structural genes, and
their protein products activate the transcription in the presence of their cognate
inducer molecule. Repressor-mediated regulation is rare for genes involved in
the catabolism of aromatic compounds. However, Mouz et al. reported that
the expression of bph genes on transposon Tn4371 was repressed by the product
of bphS gene (9), although the molecular events in the repression and
derepression have fully remained to be elucidated.
The bph genes and their organization in KKS102 are highly homologous
to the bph genes on transposon Tn4371 (10) (11). These two bph gene clusters
share 94% identity at the nucleotide level, but DNA sequences in the upstream
region of bphE are different from each other (10).
The bph genes in KKS102 are induced when grown in the presence of
biphenyl. This induction requires an inducer molecule, 2-hydroxy-6-oxo-6-
phenylhexa-2,4-dienoic acid (HOPDA), an intermediate metabolite of the
biphenyl degradation pathway (12). In this study, we identified a negative
regulator of bph genes and revealed its function in the regulation of bph gene
expression in KKS102. This report describes for the first time the detailed
regulatory mechanism of PCB / biphenyl degradation genes.
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Experimental Procedures
Bacterial strains and growth conditions
Pseudomonas sp. KKS102 (4) and its derivatives were cultivated in 1/3 L
broth (0.33% trypton, 0.16% yeast extract, 0.5% NaCl) at 30°C. Eschrichia
coli cells were grown in L broth (1% trypton, 0.5% yeast extract, 0.5% NaCl)
at 37°C. Antibiotics were used at a final concentration of 50 µg/ml for
ampicillin and 25 µg/ml for kanamycin and chloramphenicol.
Construction of the bphS disruptant
For the construction of a plasmid for bphS disruption, plasmid pKH1004
carrying a 1.2-kb HincII fragment in the multi cloning site of pUC19 was
digested by XhoI, and a chloramphenicol resistance gene derived from
pHSG399 was inserted into the cleaved site. The resulting plasmid was
linearized by BamHI and HindIII digestion and used for electroporation. The
gene disruption was confirmed by Southern blot analysis. The Southern blot
analysis was performed with an ECL gene detection system (Amersham,
Pharmacia) according to the provided protocol.
Construction of strains for LacZ reporter assay
All of the fusion constructs of the modified upstream region of bphE and
lacZ were integrated into the genome of KKS102. For systematic construction
of plasmids for integration, plasmid pKLZ-A was constructed. pKLZ-A
contains the following DNA fragments: kanamycin resistance gene derived
from Tn5 as a marker for integration, a synthetic terminator sequence to
prevent read-through of transcription, a multi cloning site comprised of EcoRI,
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SmaI, and BamHI sites and lacZ gene derived from pMC1403 (13), and these
are inserted into a randomly selected DNA fragments from KKS102 genome.
The modified promoter-lacZ fusions were constructed by inserting either
duplex oligonucleotides or PCR amplified DNA fragments into the multi-
cloning site of pKLZ-A or of its derivative plasmid. The DNA sequences
inserted are presented in Fig. 5.
For integration into chromosome of KKS strains, resulting plasmids were
digested by HindIII within vector sequence and introduced into KKS102 by
electroporation. Integration of promoter-reporter constructs into the genome
results in disruption of a single open reading frame (ORF) that encodes a
putative member of the NtrC family regulator. This disruption had no effect
on the expression level of bph genes under any conditions tested.
Construction of the pE promoter deleted mutant
For the deletion of the chromosomal pE promoter, plasmid pKH966A
was constructed. The plasmid pKH966A carries the following DNA fragments
in the cloning site of pHSG399 (see Fig. 8); a bphE upstream region (-400 to -
1284 where +1 is the start codon for bphE) , a DNA fragment derived from
pKLZ-A containing the kanamycin resistance gene and a terminator sequence,
and a bphE upstream region (-242 to +3 where +1 is the start codon for bphE).
The plasmid pKH966A was digested by HindIII and BamHI in a vector sequence
and used for electroporation.
Electroporation
Each of the plasmids was linearized by an appropriate restriction enzyme,
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extracted with phenol/chloroform, ethanol-precipitated, dissolved in water, and
introduced into KKS102 by electroporation. The cells from liquid culture
were washed five times with chilled sterile water. The Gene Pulser (Bio-Rad,
Heercules, Calif.) was used under the following conditions: 0.1-cm cuvette, 200
Ω, 25 µF, 1.8 kV, and a pulse time of 4.7 to 5.0 ms. A 1 ml aliquot of SOC
medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 10 mM MgCl2 and 20
mM glucose, pH 7.0) was added immediately after electric pulse. The cells
were incubated at 30°C for 3 hours prior to plating onto 1/3 L broth containing
appropriate antibiotics.
Northern blot analysis
The total RNA was isolated by the method described by Hopwood et al.
(14). Hybridization and detection were performed by using digoxigenin-
labeled DNA with a CSPD system (Boehringer-Mannheim, Germany),
according to the provided protocol. The 1.0-kb HincII-ApaI fragment, 1.2-kb
SphI-SmaI fragment, and 0.9-kb EcoT22I-KpnI fragment were used to generate
bphA1, bphC, and bphE probe, respectively (see Fig. 1B).
Assay for BphD activity
To measure the BphD activity, the cells were washed once in sample
buffer (50 mM potassium phosphate buffer (pH 8.0) containing 10 % glycerol)
and resuspended in 1 ml of the same buffer. After sonication and
centrifugation at 15,000 rpm for 10 min, the supernatant (crude extract) was
assayed for BphD activity.
For the assay of BphD activity, BphD substrate (HOPDA) was diluted to
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an OD 434 of 0.5 to 1.5 in the sample buffer. HOPDA was prepared as
described (12). The crude extract was added to HOPDA solution prewarmed
at 30°C. The decrease in OD 434 was measured for 3 minutes, and the portion
in which OD 434 decreased in proportion to time was used to calculate the BphD
activity. We defined 1U of BphD activity as the activity necessary to convert 1
nmol of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid per minute. A molar
extinction coefficient of 19800 for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic
acid was used to calculate the BphD activity (15). The amount of protein in the
crude extract was quantified using a protein assay kit (Bio-Rad, Heercules,
Calif.).
Assay for LacZ activity
For LacZ activity measurement, crude extract was prepared as described
above except that Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1
mM MgSO4, 50 mM β-mercaptoethanol) was used. The crude extract was
added to the ONPG solution (4 mg/ml in Z buffer). After 10 min of
incubation at 30°C, stop solution (1 M Na2CO3) was added to terminate the
reaction and O.D. 420 was measured. We defined 1U of LacZ activity as the
activity necessary to produce 1 nmol of ONP a minute.
Primer extension
The total RNA was isolated by the method described by Hopwood et al.
(14) from KKS102 cells after 3 hours of incubation with biphenyl. The
primer extension was performed with oligonucleotides BPHE10-29 (5'-
CTGGTCGAAACCGTATCTGG-3') (hybridizing to nucleotides 10 to 29 of the
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bphE coding sequence). The primer was annealed to approximately 20 µg of
the isolated RNA. Primer extension reactions with avian myeloblastosis virus
reverse transcriptase (Promega Corp.) were performed at 37°C and extended
product was run beside of the DNA sequencing ladder generated by the
dideoxy-chain termination method using the same primer. Samples were run
with a LI-COR model 4000L DNA sequencing system (LI-COR, Lincoln,
Neb.).
Nucleotide sequence determination
The nucleotide sequence was determined by the dideoxy-chain
termination method with the Applied Biosystems model 310 DNA sequencing
system (Applied Biosystems, Foster City, Calif.).
Gel retardation assay
A 145-bp fragment that contains a pE promoter region was amplified by
PCR with primers BPHUP12-BAM (5'-
GGCGGATCCGAGTGAAGTGAGTGAAAC-3') and BPHUP-REV1-SAL (5'-
GGCGTCGACCATGATTGCCCCTGCGCG-3'). BPHUP12-BAM and
BPHUP-REV1-SAL were designed to introduce a restriction site for BamHI
and SalI, respectively. The PCR fragment was digested with BamHI and SalI
and inserted into the cloning site of pYO2 (pHSG398 derivative harbouring a
multi cloning site consisting of a restriction site in the order of EcoRI, BamHI,
PstI, SalI, BglII, XbaI, XhoI, and HindIII), resulting in pYO12R. A 183-bp
HindIII-EcoRI fragment prepared from pYO12R was end labeled by [γ-32P]ATP.
The reaction mixtures (10 µl) contained 10 mM Tris-HCl (pH 7.5), 50 mM
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NaCl, 0.5 mM dithiothreitol (DTT), 10% glycerol, 0.05% NP-40, 1 µg of
poly-(dI-dC), 1 ng of 32P-labeled DNA, and cell free extract (CFE) containing 0
to 5 µg of protein. In competition assay, 40 ng of cold target DNA or 1 µg of
unrelated DNA (sermon sperm DNA) was added. After incubation for 10 min
at room temperature, the mixtures were separated by electrophoresis at 60 V
constant voltage in 5% polyacrylamide gels buffered with 1xTBE buffer.
For preparation of CFE, E. coli harboring plasmid pKH701, which
carries the bphS gene under the control of the lac promoter were cultivated in L
broth. When turbidity reached a value of 0.5, isopropylthio-β-D-galactoside
(IPTG) was added at a final concentration of 1 mM. After 5 hours of
additional incubation, cells were collected, washed once with 50 mM potassium
phosphate buffer (pH 7.4), resuspended in the same buffer, and disrupted by
sonication. After centrifugation of the sonicated cell suspension, the
supernatant was used as CFE. The protein concentration in the CFE was
determined by using the Bio-Rad protein assay kit. Bovine serum albumin was
used as a standard.
DNase I footprinting
A 183-bp fragment used for gel retardation with both ends labeled with
32P was used after the following procedures were performed. The fragment
was digested by either BamHI or XhoI. In either digestion, 177-bp and 6-bp
fragments were generated. The longer fragments, digested by BamHI and
XhoI had 32P in the coding and non-coding strand, respectively. Two rounds
of ethanol precipitation removed the shorter fragment. The single end labeled
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fragments were incubated with CFE prepared from E. coli cells expressing
BphS protein in the same buffer conditions as were present for the gel
retardation assay. The total volume of the reaction mixture was 40 µl, and 0 to
20 µg of protein in CFE was used. After 10 min incubation at room
temperature, DNase I solution (diluted in 10 mM MgCl2 5 mM CaCl2) was
added and incubated at 30°C for 2 min. The reaction was stopped by addition
of 40 µl of phenol, followed by vortexing and addition of 100 µl of stop
solution. Protected bands were identified by comparison with the migration of
the same fragment treated for A+G sequencing reactions by the method of
Maxam and Gilbert (16).
Results
Nucleotide sequence of the upstream region of bphE
The upstream region of bphE, the first gene of the bph gene cluster, was
sequenced for about 3-kb. Two ORFs were found in this region in an
orientation opposite that of the bph genes cluster (Fig. 1B). The start codon of
the ORF proximal to bphE was 511 bp distant from that of bphE. Their
nucleotide and deduced amino acid sequences are shown in Fig. 2. The
deduced amino acid sequence of the ORF proximal to bphE showed homology
to transposases of several transposons. These are IS1405 from Ralstonia
solanacearum (accession no. AAD49338), IS1384 from Pseudomonas putida
(accession no. AAC98743), and ISPSMC from Pseudomonas syringae
(accession no. BAA75460). Identities between this ORF and their transposases
are 74%, 64%, and 62%, respectively. Typical terminal 4-bp direct repeats
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and 16-bp inverted repeats flank this ORF. Because these are common
features of an insertion sequence, we designated this region and the transposase
ORF as ISBPH and tnpBPH, respectively. Southern blot analysis using a
1374-bp SphI fragment (Fig. 1B) as a probe revealed that this insertion
sequence existed as a single copy in the genome of KKS102 (data not shown).
The deduced amino acid sequence of another ORF distal from bphE
showed homology to transcriptional repressors of the GntR family (17), BphS
of transposon Tn4371 (9) and AphS of Comamonas testosteroni (18). This
ORF had 74.4% and 37.0% identities to these repressors, respectively. The
sequence also showed 40.2% identity to ORF0 from Pseudomonas
pseudoalcaligenes KF707, which also belongs to the GntR family, but
exceptionally works as a positive regulator (19). Because the product of this
ORF functioned as a regulator of the bph genes (see below), we designated it as
bphS.
Repressive function of the bphS gene product in KKS102
The bphS gene of KKS102 was disrupted as indicated in Fig. 3A (for
details see Experimental Procedures), and we analyzed the effects of disruption
on the expression of the bph genes. The strains were grown in liquid culture
with or without biphenyl and their BphD activities were measured at 1, 3, 5,
and 7 hours after addition of biphenyl. In the wild type KKS102, BphD
activity was kept at a low level in the absence of biphenyl, whereas it gradually
increased in the presence of biphenyl and reached a level of activity 5-fold that
of the original level after 5 hours. In the bphS disruptant (KKS∆S), a high
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level of BphD activity was detected even in the absence of biphenyl (Fig. 3B).
We also measured bph mRNA level in the bphS disruptant by Northern
blot analysis. Strains were incubated with or without biphenyl before mRNA
preparation. mRNAs were blotted onto nitrocellulose membranes and were
hybridized with bphA1-, bphC-, or bphE- specific probe. Signals on the
membranes were quantitatively analyzed and the amounts of bph mRNAs that
were detected with the above probes were compared with those of uninduced
wild type strain (Fig. 3C). In the wild type strain KKS102, the bph mRNA
level was high in the presence of biphenyl but not in its absence. In contrast, a
high level of bph mRNA was detected in the bphS disruptant, even in the
absence of biphenyl.
To rule out the possible polar effect of the disruption of bphS with a
chloramphenicol resistance gene on the expression of bph genes, a fusion
construct of the bphE upstream region and lacZ as a reporter gene(Fig. 3D) was
integrated into a randomly selected site on the KKS102 genome (see
Experimental procedures), and the effect of bphS disruption was analyzed. In
the bphS+ strain (KLZ12), LacZ activity was about 3 times higher in the
presence of biphenyl than in its absence. In contrast, we detected a high level
of LacZ activity in the bphS disruptant (KLZ12∆S) in the absence of biphenyl,
and this level was even higher than that in the presence of biphenyl. These
results clearly demonstrate that the bphS gene product negatively regulates bph
gene expression in KKS102.
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Characterization of a promoter located upstream of bphE
Because the bphE gene, the first gene in the bph gene cluster, is induced
by biphenyl, we searched for a promoter in the upstream region of bphE. To
identify the transcription start site in vivo, primer extension analysis was
performed. When the primer BPHE10-29, which hybridized to the
nucleotides 10 to 29 of the BphE-coding sequence, was used, a single band
corresponding to a T residue located at 317 nucleotides upstream from the bphE
start codon was detected (Fig. 4). The transcription start site was preceded by
a conserved E. coli –10 box (TATAAT). The -35 box was not highly
consistent with that of E. coli (GTGTTT Vs TTGACA of E. coli). The
location of these elements was in good agreement with the results from LacZ
reporter assay (see below). No other transcriptional start site was identified.
Hereafter, we refer to the DNA region that includes the -10 and -35 boxes
(-326 to -354 where +1 is the translation start point for bphE) as the pE core
promoter, and the DNA region that includes the pE core promoter and the other
elements involved in the transcriptional regulation as the pE promoter.
Identification of the DNA region required for inducible expression
of the pE promoter
We performed a deletion analysis of the bphE upstream region to define
the sequence necessary for inducible promoter activity. A series of fusion
constructs of the partially-deleted 5' region of the bphE and lacZ gene was
integrated into the genome of KKS102 (see Experimental Procedures for
details), and LacZ activity was measured in the presence or absence of biphenyl.
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By integrating the lacZ fusion constructs into the genome, the effect of
difference in copy number of the reporter gene could be excluded from the
measurement of the promoter activity. The results are summarized in Fig. 5A.
First, pE is the sole promoter that resides within 1555 bp upstream of the bphE
start codon. The same level of LacZ activity was observed in strain KLZ10
(containing up to 1555 bp from the bphE translation start site) and in strain
KLZ12 (containing up to 387 bp from the bphE translation start site), indicating
that there is no promoter activity between nucleotides –388 and -1555. The
deletion of the pE core promoter results in a markedly low level of LacZ
activity (compare strains KLZ12 and KLZ9). Deletion of the sequence just
upstream of the –35 box results in a decrease in LacZ activity (see strains
KLZ14 and KLZ8). This decrease may be due to deletion of the UP element,
the third DNA element of the prokaryotic core promoter (20). Second, the
DNA region from -387 to -243 (where +1 is the bphE translation start site) is
sufficient and necessary for promoter activity and the inducible expression,
because strain KLZ23 (which contains –387 to –243) showed enhanced LacZ
activity that was much higher in the presence of biphenyl.
Specific binding of BphS to the pE promoter
The results described above suggested that the bphS gene product binds
within the DNA region spanning from nucleotides -387 to -243. In order to
determine whether the BphS binds to this DNA region, we conducted gel
retardation experiments. The DNA region from -387 to -243 of the pE
promoter was excised as an EcoRI-HindIII fragment from pYO12R and was end
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labeled with 32P (Fig. 6A). The BphS protein was expressed in E. coli, and the
cell free extract (CFE) was used for gel retardation assay. The retarded bands
were observed only in the reactions containing BphS protein (Fig. 6B). There
were two shifted bands at higher protein concentrations, suggesting that BphS
binds to multiple sites within the fragment. When the nonradioactive DNA
fragment was added to the binding reaction in excess of the 32P-labeled fragment,
the retarded band was greatly reduced (Fig. 6B, lane 6). The addition of an
unrelated DNA fragment did not affect the BphS binding (Fig. 6B, lane 7).
The retarded band was not observed when CFE of E. coli harboring vector
pHSG399 was used (Fig. 6B, lane 8). These results clearly indicate that BphS
specifically binds to the DNA region spanning -387 to -243.
Inhibition of binding of BphS to the pE promoter by HOPDA
In our previous work, we demonstrated that HOPDA, the intermediate
metabolite of the biphenyl degradation pathway, is the inducer molecule of bph
genes in KKS102 (12). We here performed a gel retardation assay in the
presence of varying concentrations of HOPDA (0 to 0.5 mM). The amount of
protein of CFE was fixed at 3 µg. Under this condition only one retarded band
was observed (see Fig. 6B, lane 3). As shown in Fig. 6C, the intensity of the
retarded band was reduced in a concentration-dependent manner. In the
presence of HOPDA at 0.5 mM, the retarded band disappeared almost
completely (Fig. 6C, lane 6). In contrast, a saturated concentration of
biphenyl (approximately 0.1 mM) or 0.5 mM 2,3-dihydroxybiphenyl did not
inhibit the binding. These results indicate that the BphS protein loses its ability
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to bind to the pE promoter in the presence of the inducer molecule, HOPDA.
This inhibition of binding of BphS to the pE promoter by HOPDA is consistent
with the in vivo function of HOPDA as an inducer.
DNase I footprinting analysis
To obtain further information on the binding site of the BphS protein,
DNase I footprinting analysis was carried out. A 183-bp pE promoter
fragment containing the region from -387 to -243 was analyzed with both
coding and non-coding strands (Fig. 7). The results of the DNase I
footprinting are summarized in Fig. 7B. We identified four BphS binding
sites and named them (beginning with the furthest upstream) BS I (binding site
I), BS II, BS III, and BS IV. When DNA labeled in the coding strand was
incubated with a relatively low amount of CFE containing BphS, two regions
ranging from nucleotide -333 to -319 (BS I) and -315 to -299 (BS II) were
protected from DNase I digestion (lane 3). When DNA labeled in the
noncoding strand was used under the same protein and DNA concentrations,
DNA regions ranging from -329 to -315 (BS I) and -312 to -295 (BS II) were
protected (lane 8). At higher protein concentrations, additional DNA regions
from -296 to -281 (BS III) and -279 to -263 (BS IV) (lane 5, analyzing coding
strand), and from -291 to -278 (BS III) and -274 to -260 (BS IV) (lane 10,
analyzing noncoding strand) were protected. In Fig. 7, the DNA regions
protected at low and high protein concentrations are indicated by thick and thin
lines, respectively. No protection was observed when CFE of E. coli
harboring vector plasmid was used (data not shown). These results
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demonstrate that the BphS protein binds to four sites near the pE core promoter
and has greater affinity to the two upstream binding sites (BS I and BS II) than
the two downstream sites (BS III and BS IV). That a part of -10 box hexamer
of the pE promoter overlaps with BS I and was protected from DNase digestion
suggests the essential role of BS I in BphS-mediated repression of the pE
promoter.
In vivo function of the BphS binding sites
The results of the DNase footprinting demonstrated several interesting
properties of the four binding sites. The protection of BS I and BS II, but not
BS III and BS IV, under low protein concentration of BphS-containing CFE
indicates that BS I and BS II have greater affinity for BphS protein than do BS
III and BS IV.
To investigate the function of these two sets of BphS binding sites in the
repression of the pE promoter in vivo, a series of promoter constructs was
integrated into the genome of KKS102 as described in the previous sections and
assayed for promoter activity (Fig. 5B). The strain KLZ23, which had a
construct with all four binding sites as well as the pE core promoter, showed
low LacZ activity when grown in the absence of biphenyl. In contrast, the
strain KLZ22, the integrated construct of which had BS I and BS II but lacked
BS III and BS IV, showed high LacZ activity even in the absence of biphenyl.
The LacZ activity in the absence of biphenyl was increased approximately 3-
fold, although it did not exceed that in the presence of biphenyl, by deletion of
BS III and BS IV (compare strains KLZ23 and KLZ22 in the absence of
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biphenyl), demonstrating the in vivo function of BS III and BS IV. In strain
KLZ21, where the integrated construct had no BphS binding site, LacZ activity
in the absence of biphenyl was further elevated, to the level observed in the
strain KLZ15, whose integrated construct lacked BSI and BS II but had BS III
and BS IV. This result indicates that the presence of BS I and BS II is essential
for repression of the pE promoter.
In the strains KLZ12∆S, KLZ21, and KLZ15, the presence of biphenyl
resulted in lower lacZ activities than its absence. This might have been due to
the cytotoxicity of biphenyl (21) and / or the catabolite-repressive effect on the
pE promoter as a result of biphenyl assimilation.
In conclusion, BS I and BS II were found to play an essential role in
repression in vivo, and another set of two binding sites was shown to be
functional, although its role was auxiliary.
Role of the pE promoter in the expression of entire bph genes
The results presented above demonstrate the role of BphS in the
regulation of the pE promoter. In a previous section, we have demonstrated
that disruption of the bphS gene resulted in constitutive expression of the bphA1,
bphC, and bphE genes (Fig. 3). This suggests that BphS plays a central role in
the regulation of entire bph genes and raises the following questions. Does the
pE promoter drive the transcription of the entire bph gene cluster? Are there
any other BphS-regulated promoters in the bph gene cluster? To address these
questions, the pE promoter region from -242 to -400 in the strain KKS102 was
replaced with a kanamycin resistance gene and a transcription terminator as
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depicted in Fig. 8. Since the absence of the pE promoter could hinder the
accumulation of the inducer molecules and could lead to persistent repression of
any promoters in the bph genes cluster by BphS, the effect of deletion of the pE
promoter was also tested in a strain with a bphS disrupted background. The
resulting KKS102 and KKS∆S derivatives were designated as KKS∆pE and
KKS∆pE∆S, respectively. We then investigated the induction of BphD activity
(Table I). The bphD gene is located relatively downstream in the bph genes
cluster, and therefore BphD activity serves as an indicator of the presence of
any intervening promoters. The BphD activities in the KKS∆pE were very
low irrespective of the presence or absence of biphenyl, even lower than that of
the uninduced wild type strain. In addition, the bph mRNA level detected by the
bphA1-, bphA4-, bphC-, or bphD-specific probe was low and no induction by
biphenyl was observed. We thus concluded that pE is the primary promoter
for the transcription of most bph genes.
Discussion
The bphS gene product is a negative regulator of bph genes
In the bphS gene disruptant, expression of BphD and LacZ reporter
activity, as well as amounts of bph gene transcripts, were elevated even in the
absence of biphenyl to the level of those in the induced wild type strain,
indicating that the bphS gene product plays an essential role in repression of the
bph genes. This repression results from the direct action of the bphS gene
product at the pE promoter, because the BphS protein specifically bound to the
pE promoter and deletion of BphS binding sites led to constitutive production of
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LacZ activity. The repressor function of BphS is consistent with the fact that
BphS belongs to a GntR family of transcriptional repressors.
pE promoter, an essential promoter for bph genes transcription
The LacZ reporter assay of the upstream region of bphE revealed that
only one promoter, which was designated the pE promoter, exists in the region.
Elimination of the pE promoter resulted in weak and constitutive production of
BphD activity, as well as of bphA1, bphA4, bphC, and bphD transcripts,
demonstrating that the pE promoter plays an essential role in bph genes
expression and that bph genes (at least from bphE to bphA4) constitute an
operon. Although some other parts of the nucleotide sequence may exert
promoter activity in the long (12kb) bph gene cluster, it seems that such
promoter activities are trivial compared to the promoter activity of the pE
promoter. For example, we detected promoter activity upstream of the bphA1
gene, but fusion with the lacZ gene showed that the activity was significantly
lower (20 times lower) than that observed for the pE promoter (data not shown).
In conclusion, the pE promoter is the primary promoter driving transcription
of bph operon.
BphS binding to the pE promoter
In vivo and in vitro experiments showed specific binding of BphS protein
to the pE promoter, indicating that the BphS protein plays an essential role in
regulation of the bph operon, because the pE promoter is the primary promoter
involved in expression of the bph operon.
In order to identify additional binding sites for BphS protein that might
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be involved in repression, we performed a gel retardation assay. We used
various DNA fragments derived from the bphE upstream region as well as the
bphE-coding region (from -2262 to +396, where +1 represents the bphE
translation start codon), but we did not identify any additional BphS binding
sites (data not shown).
Consensus operator sequence of BphS protein
DNase footprinting analysis identified four binding sites for BphS just
downstream of the pE promoter. The binding sites were named, beginning
with the most upstream site, BS I, BS II, BS III, and BS IV (Fig. 7B). The gel
retardation assay revealed that the affinity of BphS to these sites is greater in the
order of BS II > BS I >BS IV >BS III (unpublished data). We also tested an
inverted repeat sequence found in the vicinity of the promoter of the bph genes
on Tn4371 (9) (Fig. 9) and found that BphS of KKS 102 bound to this sequence
at affinity greater than to BS II (unpublished data). The operator sequences
for GntR family members have been suggested to contain perfect or imperfect
inverted repeat sequences (22). BS I contains an imperfect inverted repeat
sequence and BS II contains a perfect inverted repeat sequence, while BS III and
BS IV have no distinct inverted repeat sequence. Alignment of the three
stronger binding sites with distinct inverted repeat sequences (BS I, BS II, and
the inverted repeat sequence found in Tn4371) identified the consensus sequence
of AN12T (Fig. 9) in an AT-rich symmetric sequence, which was reminiscent of
the binding motif (TN11A) for LysR-type transcriptional regulators (23).
Conservation of A and T nucleotides separated by 12 base pairs seems important,
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based on the fact that, in several DNA binding proteins which use a helix turn
helix motif for binding, two recognition helixes in the dimers are separated
about one turn of DNA helix long (24). In the DNA sequence of BS III and BS
IV, the AN12T motifs were found, although these binding sites were less
symmetric, which might be reflected in the comparably weak binding affinity
for BphS (Fig. 7B). Further investigations will be needed to clarify the
importance of the AN12T motif of BphS binding sites.
The AT content around the binding site was 78.4% (in the 74 nucleotides
from -333 to -260), and this value was very high for the bph genes, in which the
average GC content was 62%. The abundance of AT base pairs may have
helped to form a structure favored by BphS binding.
Repression mechanism mediated by four BphS binding sites and BphS
protein
The data obtained from promoter-lacZ fusion indicates that the proximal
two binding sites (BS I and BS II), as well as the distal sites (BS III and BS IV),
are functional in vivo for repression, and that BS I and BS II play a primary
role in repression. How, then, do these four binding sites and the BphS protein
mediate transcriptional repression? Protection from DNase I cleavage of part
of the -10 box by BphS indicates that the BphS protein bound at BS I masks the
-10 box of the pE promoter and prevents the access of RNA polymerase as has
been described in many other cases, as for example, the binding of the phage λ
cI repressor to the operators OR1 and OR2 (25) and also the binding of LacI to the
O1 operator of the lac promoter (26). The role of BphS proteins bound at BS
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II may be to enhance repression through stabilization of BphS binding to BS I
through protein-protein interaction. BphS bound to BS I may also stabilize
binding of BphS to BS II, and thus the binding of BphS repressors to these sites
could be cooperative. In support of this possibility, only two spices of
retarded band were observed in the gel retardation assay; one might represent
binding to BS I and BS II and the other, additional binding to BS III and BS IV,
suggesting preferable binding of BphS protein to each set of operator sites. In
addition, the finding of simultaneous protection of BS I and BS II and of BS III
and BS IV in the DNase footprinting analysis was consistent with the notion that
the BphS protein has a cooperative binding property. In our recent study,
purified 6xHis tagged BphS protein was shown to bind to a DNA fragment
containing both BS I and BS II with 10 times more efficiency than to a DNA
fragment containing only BS II (as mentioned in the preceding section, the
affinity for the BphS protein is stronger than that of BS I), supporting the
cooperative binding of BphS to BS I and BS II (unpublished data). In a recent
review on bacterial transcriptional regulation, possible cooperative interaction
of GntR dimer pairs was suggested because the candidate binding sites for GntR
occured in pairs in several cases (27). A GntR family protein, AphS from
Comamonas testosteroni TA441, binds to two sites in the promoter region,
although the implications of the presence of these two binding sites are not
known (18).
It has been reported that multiple binding sites for other types of negative
regulators are necessary for efficient repression. For example two GalR
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dimers bound to two operators have been shown to interact with each other and
repress transcription of the gal operon (28). And LacI binds to three
operators and cooperates in repression (29).
Derepression of BphS mediated repression by HOPDA
We demonstrated in vitro that BphS protein binds specifically to the pE
promoter, and that the binding affinity decreases in the presence of HOPDA.
This feature of the BphS protein enables high levels of the expression of bph
genes only when the intermediate of the biphenyl degradation pathway is
present. This finding is well consistent with the previous finding that HOPDA
is the inducer of bph genes in vivo (12).
BphS protein, a key regulator of bph gene transcription
The question of the mechanisms by which gene expression occurs only
under particular circumstances is of great interest. A model depicting the
molecular aspect of bph gene regulation is as follows. In KKS102 cells, the
BphS protein binds to its binding sites and inhibits transcription from the pE
promoter. In this repressed state, binding of BphS to BS I plays a central role
and BphS protein bound to the other sites helps to stabilize the BphS protein
bound at BS I. When the cells encounter biphenyl, biphenyl is converted to
HOPDA by bph gene products that are somehow maintained at the basal level,
leading to dissociation of the BphS protein from the operator DNA and to
subsequent active transcription initiation at the pE promoter. The increase in
bph gene products results in further elevation of HOPDA concentration. In
conclusion, the BphS protein is a key component of the molecular switch
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regulating expression of the bph operon in KKS102. It regulates the pE
promoter that is essential for expression of the bph operon.
Acknowledgments
Y. O. is financially supported by research fellowships of the Japan Society for
the Promotion of Science for Young Scientists. This work was supported in
part by a Grant-in-Aid from the Ministry of Education, Science, Sports, and
Culture of Japan. This work was performed using the facilities of the
Biotechnology Research Center, The University of Tokyo.
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Embo J 9(4), 973-9
Footnotes
The nucleotide sequence data reported in this paper have been deposited
in the DDBJ nucleotide sequence database under accession no. AB047327 for
tnpBPH and AB047328 for bphS.
The abbreviations used are: HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-
2,4-dienoic acid; PCB, polychlorinated biphenyl; CFE, cell free extract; BS,
binding site
Figure Legends
Fig. 1. A. The PCB / biphenyl degradation pathway in
Pseudomonas sp. KKS102. Biphenyl is converted to dihydrodiol
compound by BphA activity (1). The dihydrodiol compound is converted to
2,3-dihydroxybiphenyl (1) by BphB activity. 2,3-dihydroxybiphenyl is
converted to 2-hydroxy-6-oxo-6-phenyhexa-2,4-dienoic acid (HOPDA) by meta
cleavage activity exerted by BphC (4). BphD is a hydrolase and converts
HOPDA to two molecules, benzoic acid and 2-hydroxypenta-2,4-dienoate (4).
Further, 2-hydroxypenta-2,4-dienoate is converted to acetyl-CoA and pyruvate
by 2-hydroxypenta-2,4-dienoate hydratase (BphE), 4-hydroxy-2-oxovalerate
aldolase (BphF), and acetaldehyde dehydrogenase (BphG) (5). B. The bph
gene cluster in KKS102. The two ORFs found in this study are also shown.
Lines under the gene cluster represent the DNA fragments used to generate
probes for Northern and Southern blot analyses.
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Fig. 2. The nucleotide sequence of the upstream region of the bph
gene cluster. The deduced amino acid sequences of tnpBPH and bphS are
shown. Asterisks indicate the stop codons. The 4-bp terminal direct repeats
and 16-bp terminal inverted repeats flanking tnpBPH are indicated. The
putative ribosome binding site of bphS is underlined. The predicted helix turn
helix DNA binding motif in the BphS amino acid sequence is shown by white
letters under black background. The ATG start codon (underlined) of the
divergently transcribed bphE gene is located at nucleotides 1 to 3.
Fig. 3. Characterization of the bphS disruptant. A. Schematic
representation of the strategy for bphS disruption. The bphS gene was
disrupted by inserting chloramphenicol resistance gene by a double crossover
homologous recombination. B. BphD activity in the bphS disruptant.
KKS102 (circle) and the bphS disruptant (square) were incubated in 100 ml 1/3
L broth. When turbidity at O.D. 660 reached a value of 0.5, the culture was
divided into halves, and further incubated in the presence (closed symbol) or
absence (open symbol) of biphenyl. The BphD activity was measured after 1,
3, 5, and 7 hours of incubation. C. The expression of the bph gene
transcripts in the bphS disruptant. KKS102 and the bphS disruptant were
incubated in 100 ml 1/3 L broth. When turbidity at O.D. 660 reached a value of
0.5, the culture was divided into halves and further incubated in the presence or
absence of biphenyl. After 3 hours of additional incubation, cells were
harvested and the total RNAs were prepared. RNA samples were slot-bloted
onto the nitro cellulose membrane and probed with bphA1, bphC, and bphE.
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The results of Northern blot analysis were quantitatively analyzed. The
relative amounts of the mRNA are shown. D. LacZ activity in the strain
harbouring chromosomally integrated bphE promoter-lacZ transcriptional
fusion. The bphE upstream region up to position -387 (+1 represents the bphE
start codon) was fused with the lacZ gene and integrated into the genome of
KKS102 (strain KLZ12). KLZ12 and its bphS disruptant derivative,
KLZ12∆S, were incubated in 1/3 L broth and when turbidity at O.D. 660 reached
a value of 0.3, the culture was divided into halves and further incubated in the
presence or absence of biphenyl. After 6 hours, the cells were harvested for
LacZ activity measurement.
Fig. 4. Primer extension analysis of RNA from KKS102 cells. RNA
was purified from cells of cultures grown in 1/3 L broth supplemented with
biphenyl. On the right is the sequence pattern of a dideoxynucleotide
sequencing reaction. The arrows indicate the primer extension product and
the proposed start site of transcription in the sequence at the left. The
nucleotide sequence of the bphE promoter region is shown below, with the -10
and -35 box underlined.
Fig. 5. Deletion analysis of bphE upstream region.
LacZ activity in a series of strains harboring fusion of 5' region of bphE and
lacZ gene in the chromosome. The DNA region fused with lacZ is shown with
white bar at the left. The numbers in the figure represent the marginal
nucleotide positions of the fusion constructs (+1 represents the bphE translation
start codon). Each strain was incubated in 1/3 LB and LacZ activity was
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measured in the same conditions as described in Fig. 3D. LacZ activity in the
cells incubated with (filled bars) or without (empty bars) biphenyl is shown on
the right. In panel B, nucleotide sequence around the pE promoter is shown.
Putative BphS recognition sequences deduced from DNase I footprinting are
boxed. The four binding sites are also denoted in the figure by black boxes.
Fig. 6. Specific binding of the BphS protein to the promoter region.
A. Schematic diagram of the plasmid used to generate the probes for gel
retardation assay and DNase I footprinting. DNA region -387 to -243 (+1
represents the bphE translation start codon) was amplified by PCR and cloned
into the BamHI and SalI site of plasmid pYO2. B. Gel retardation assay
demonstrating the specific binding of BphS to the DNA region. 1 ng of
labeled fragment was incubated with no protein (lane 1), CFE of E. coli
harboring pKH701 (a plasmid coding bphS, lanes 2-7) or CFE of E. coli
harboring pHSG399 (vector for pKH701) (lane 8). Protein concentration were
(in µg per 10 µl reaction mixture): 0.05 (lane 2); 0.3 (lanes 3, 6, and 7); 1.0
(lane 4); 5.0 (lanes 5 and 8). 40 fold excess of the unlabeled fragment (lane 6)
or 1 µg of unrelated fragment (sermon sperm DNA) (lane 7) was added to the
mixture. Unbound free DNA is marked (F); bound DNA is marked C1 or C2.
C. Binding of BphS in the presence of the inducer molecule. 1 ng of labeled
fragment was incubated with 0.3 µg of CFE of E. coli harboring pKH701.
Lanes 1-6 contain the following concentration of HOPDA: 0, 5, 25, 50, 250,
500 µM. Lane 7 contains saturated concentration of biphenyl (approximately
0.1 mM). Lane 8 contains 2,3-dihydroxybiphenyl (500 µM).
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Fig. 7. DNase I footprinting of BphS binding to the pE promoter
region. A. DNase I footprinting of the DNA fragment labeled for the
coding (lanes 1-5) and non-coding (lanes 6-10) strands. Lanes 1 and 6,
Maxam-Gilbert G and A reaction products. Lanes 2 and 6, DNase I digests in
the absence of CFE. Lanes 3, 4, and 5 (8, 9 and 10) DNase I digests of the
reaction mixture containing the following amount of protein in CFE of E. coli
harboring plasmid pKH701: 1 µg (lanes 3 and 8), 5 µg (lanes 4 and 9), and 20
µg (lanes 5 and 10). The thick lines indicate the regions protected from DNase
I digestion when a lower amount of CFE (1 µg) was used. The thin lines
indicate the regions protected when a higher amount of CFE (5 or 20 µg) was
used. Numbers at the left indicate nucleotide positions relative to the
translation start site of bphE. B. Summary of footprinting data of BphS
binding to the pE promoter. The protected regions are indicated by thick and
thin lines as described above.
Fig. 8. Schematic representation of the strategy for disruption of the
pE promoter. The pE promoter was replaced by a kanamycin resistance
gene and a transcriptional terminator by a double cross over homologous
recombination.
Fig. 9. Comparison of binding sites. Binding sites I, II, III, and IV, as
well as BphSKKS102 binding sequence from Tn4371 are shown. Conserved A
and T bases are shown by white letters. Bases which consist symmetry are in
bold type. Vertical line indicates the axis of symmetry.
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bphS
O
COOH
OH
Cl
O2+NADH+H+
BphE BphF BphGBphD
pyruvate NAD++CoASH NADH+H+
CH2
OH
COOH CH3
O
COOH
HO
CH3
CHO
CH3
COSC 0A
Cl
COOH
Cl
OH
OH
H
H
Cl
OH
OH
Cl
BphA1A2A3A4 BphCBphB
bphF bphC bphD bphA4 bphRbphG bphA1 bphA3bphA2 bphBbphE orf1orf4
1kb
HincII ApaIEcoT22I KpnI SphI SmaISphI SphI
tnpBPH
Insertion sequence
(A)
(B)
Y. Ohtsubo et al. Fig. 1
NAD+ NAD+ NADH+H+ O2
H2OH2O
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1 CATTCGAGATTTCTCCTTGGTTCATGGTTTCTGCGATTTCCGTGCGCACTGCCCGTCGGT 60
61 TGTGATCGCATGCATATGCGTGTGCAAGCGAAGGTCAGCCTGGCCCCTGCTTGGCTGATC 120 121 GCTCATGCAGGCACCCAAAAACTTGTGTCCCCAGATACTGATGCGGTCGTTGCGCACGAC 180 181 CGCATCAGGGAAAAGGGACTGTTGCCGAACACCGCCAAGTGATTGCATGGATGAGCACCG 240 241 AAGCGCATGATTGCCCCTGCGCGAACAATCTTTTTTTGAACATATTGTTGCCATTGATTT 300 301 TAAATAACGTTATTTTTATATAAAATAGATTATATGCATGCCATAATTTTTAAACACTCG 360 361 TGAAAACCCTATGTTTCACTCACTTCACTCCGCGCACTTGCGCCGGGCTGACACATTGAT 420 421 GCACCTCTCAACGACGACCGCGTTAGGGATGGTGTTCAAAACTCCGGTCAAGCCCGGATG 480 481 GCGTGAGCGAGTTACGACACGAACAATGGCGTCATGACCCAACAAGACCTCGGCCTGAAC 540 TpnBPH M T Q Q D L G L N 541 CTGAGCACGCGGCGTACTCGCAAAACCGTGTTCCTCGACGAGATGAACCTGGTGGTGCCG 600 L S T R R T R K T V F L D E M N L V V P 601 TGGACGGAGTTGCTGTCGTTGATTGCGCCGCATGTACCTCGCGCCAAGACCGGACGCCCG 660 W T E L L S L I A P H V P R A K T G R P 661 CCGTTTGAGCTGGTGACGATGTTGCGCATTCATTTCTTGCAACAGTGGTTCGGCCTGAGT 720 P F E L V T M L R I H F L Q Q W F G L S 721 GACCTGGCCATGGAAGAAGCCCTGTTCGAGACCACGCTGTACCGCGAGTTCGCGGGACTC 780 D L A M E E A L F E T T L Y R E F A G L 781 TCCAGCGCCGAGCGCATCCCTGACCGGGTCAGCATCCTGCGCTTTCGCCACCTGCTCGAA 840 S S A E R I P D R V S I L R F R H L L E 841 GAACACCAGTTGGCCCCGCAGATGCTGGCCGTGGTCAACGCCACCCTGGCCGACAAAGGC 900 E H Q L A P Q M L A V V N A T L A D K G 901 TTGATGCTCAGACAAGGCACGGTGGTGGACGCCACCTTGATTGCTGCGCCCAGTTCGACC 960 L M L R Q G T V V D A T L I A A P S S T 961 AAGAACCAGGATGGCAAGCGTGATCCCGAGATGCACCAGACCAAGAAGGGCAACCAGTGG 1020 K N Q D G K R D P E M H Q T K K G N Q W 1021 CATTTCGGCATGAAAGCGCACATCGGCGTGGACGCTGACTCGGGACTGGTGCACACCGTG 1080 H F G M K A H I G V D A D S G L V H T V 1081 GTCGGTACAGCAGCCAACGTCAACGACGTGACACAGGCCAGTGCGTTGGTCCATGGCGAA 1140 V G T A A N V N D V T Q A S A L V H G E 1141 GAAACGGATGTGTTCGCTGACGCCGGCTACCAGGGCGTGACCAAGCGCGAAGAAGTCCAA 1200 E T D V F A D A G Y Q G V T K R E E V Q 1201 GGCATCGATGCCAACTGGCATGTGGCCATGCGTCCGGGCAAGCGCCGCGCGATGGACAAG 1260 G I D A N W H V A M R P G K R R A M D K 1261 AACAGTCCCATGGGCGCCGTGCTCGACCAGCTTGAACACGTCAAGGCCCGAATCCGGGCC 1320 N S P M G A V L D Q L E H V K A R I R A 1321 AAGGTGGAACACCCGTTTCGGGTCATCAAACGGCAGTTCGGCCACATGAAAGTGCGCTAT 1380 K V E H P F R V I K R Q F G H M K V R Y 1381 CGGGGACTGGCCAAAAACACGGCACAGTTGCACACGCTGTTTGCACTGAGCAATCTTTGG 1440 R G L A K N T A Q L H T L F A L S N L W 1441 ATGGTGCGACGCCGACTGTTGCAAGGGCTGCAGGCGTGAGTGCGTCCGCAAGCAGCCGAA 1500 M V R R R L L Q G L Q A * 1501 GGGCCGCCGCCGAACGGAAAATGGCCTGTGAAAACGCAGAAACTGGGTCGAATTCGCCGA 1560 1561 TTCCAGGCCGCGCTGTTTCATGTCGTGGCAACCCGCCCGCTATCGGCTGGGGCCAAACGT 1620 1621 GTTTTGAACACCATCCTTAGATGTGCAACGATGTGTGACGGTTGCCGCGGCTCCGCACCG 1680 1681 GTGCGGGCTACGTTGGCCCTGCAGCACGAGCATGCAACACTATGGTGAGCACGTGGAGCG 1740 1741 GGCCTGCGGCCAGCGGCGTCACGCGGACCGGGCTGTCACGTCAGTCCTTGATCGCACAAT 1800 1801 GCTGTCTACAACCAAACAATGAAACCGAAGACCATGACAAAGCAAGATCAAGCAGTTCTG 1860
BphS M T K Q D Q A V L 1861 CCTCGATTGATTGAGTCAGCAAGACTGCCCGAAGGGGCACTGGCAGAATTCAATGTCGGG 1920 P R L I E S A R L P E G A L A E F N V G 1921 CCGAAAGAGAAGAAAGCCGTAACCGCCATCGAATCCACCTATGCAACCCTCCGTGACGCA 1980 P K E K K A V T A I E S T Y A T L R D A 1981 ATCTTGAGGGGAAGCTACCCACAGAGCTCTAGGCTCCACCTCGAGACGCTCAAGTCATCG 2040 I L R G S Y P Q S S R L H L E T L K S S 2041 CTGGGCGTCAGCGGCAGCACTCTGCGGGAAGCATTGACGCGCCTGATTGGAGACCGTCTG 2100 L G V S G S T L R E A L T R L I G D R L 2101 GTCGTCGCTGAAGGACAGAAGGGGTTCAGGGTGGCCCCAATGTCGCTTTGCGATCTTGAT 2160 V V A E G Q K G F R V A P M S L C D L D 2161 GACCTGACGAGTGCGCGCATCATGTTGGAAAGCGCCGCCCTCGTCGAAAGCATCAACCTC 2220 D L T S A R I M L E S A A L V E S I N L 2221 GGTGGGGACGGCTGGGAAGACCAACTCGTCACCAGTTTCCGTCGACTGACGAGGGCGCAG 2280 G G D G W E D Q L V T S F R R L T R A Q 2281 GAACGAGTCGAGGCCAATCCGGCAGAGGCCTTCGACGCGTGGGAAGCGAGGAACTTGGAG 2340 E R V E A N P A E A F D A W E A R N L E 2341 TTCCACAACGCACTCATGGCCGCCTCTCCGTCCAAATGGCTCGCGAACTTCAGAGAAATC 2400 F H N A L M A A S P S K W L A N F R E I 2401 CTGCTTCGAAACTCTGAGCGCTACCGCAGGTTGTCCGGCACGCAAGGCCCACTTCCTGCC 2460 L L R N S E R Y R R L S G T Q G P L P A 2461 GAAGTGCACGAGGAGCACAAGACAATCTTCGACGCGGCGATGGCCCGTGATGTAGATCGG 2520 E V H E E H K T I F D A A M A R D V D R 2521 GCGGTTTCGGCCCTCTCGCAGCATATTCGCCGCTCCGCGAATGTGATTCGAGCAAATGGA 2580 A V S A L S Q H I R R S A N V I R A N G 2581 TTGCTGAGGAAGGTCTGACCTCGACCCGTTTCCCCATGCACCGGGACCTAATGGGCTTCG 2640 L L R K V * 2641 ACTTCACCGCCTCGGTGGTGGACCCAAAGCTGGTTCAGCAACTCGCGACGCTGGAGGTCA 2700
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1kb
bphEtnpBPHbphS
XhoI
Cmr
HincII HincII(A)
(B)
Bph
D a
ctiv
ity
Uni
t / m
g pr
otei
n
(C)
1
2
3
4
5
6
7
8
9
10
bphA1
1
2
3
4
5
6
7
8
9
10
KKS102 KKS∆S
bphE
1
2
3
4
5
6
7
8
9
10
bphC
rela
tive
inte
nsity
rela
tive
inte
nsity
rela
tive
inte
nsity
biphenyl biphenyl biphenyl
tnph S
Y Oht b t l Fi 3
0
120
40
80
100
60
20
0 1 3 5 7
KKS102 KKS∆S KKS102 KKS∆S
Lac
Z a
ctiv
ity
Uni
t / m
g pr
otei
n
biphenyl
KLZ12 KLZ12∆S
250
500
750
1000
1250
1500
0
(D)
hour
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TTTTCACGAGTGTTTAAAAATTATGGCATGCATATAATCTATTTTATATAAAAAT
-35 box -10 box
A C G T
GATAAAATATATTTTTAT
+1
Y. Ohtsubo et al. Fig. 4
-317
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nloaded from
Y. Ohtsubo et al. Fig. 5.
0 500 1000 1500 2000 2500
KLZ23
KLZ11
KLZ9
KLZ8
KLZ14
KLZ12
KLZ6
KLZ5
KLZ4
KLZ3
KLZ10
biphenyl
biphenyl
-1555
-762
-648
-544
-439
-387
-370
-357
-295
-15
-243-387
-15
1600 1200 800 400 0
DNA region fused with lacZ(A)
(B)
-387
-387 -323
-290
-243
-387
-387
-387
-15
-15
-15
GAGTGAAGTGAGTGAAACATAGGGTTTTCACGAGTGTTTAAAAATTATGGCATGCATATAATCTATTTTATATAAAAATAACGTTATTTAAAATCAATGGCAACAATATGTTCAAAAAAAGATTGTTCGCGCAGGGGCAATCATG
-387 -243
BS I BS II
-35 box -10 box-357 -295 -290-323-370
BS III BS IV
KLZ23
KLZ22
KLZ21
KLZ12
KLZ15
DNA region fused with lacZ LacZ activity (unit / mg protein)400 300 200 100 0 strains
biphenylbiphenyl-295
KLZ12DS
0 500 1000 1500 2000 2500
-323
-387
strains
LacZ activity (unit / mg protein)
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EcoRIBamHI SalI BglII XbaI XhoI HindIII-387 -243
pYO12R
1 2 3 4 5 6 7 8
F
C1C2
1 2 3 4 5 6 7 8
F
C1
(A)
(B)
(C)
Y. Ohtsubo et al. Fig. 6
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1 2 3 4 5
-317-320
-330
-340
-350-360-370-380
-310
-300
-290
-280
-270
-35 box -10 boxCACGAGTGTTTAAAAATTATGGCATGCATATAATCTATTTTATATAAAAATAACGTTATTTAAAATCAATGGCAACAATATGTTCAAAAAAAGATTGTTCGCGCAGGGGCAATCATGGTGCTCACAAATTTTTAATACCGTACGTATATTAGATAAAATATATTTTTATTGCAATAAATTTTAGTTACCGTTGTTATACAAGTTTTTTTCTAACAAGCGCGTCCCCGTTAGTAC
ATAATCTATTTTAT AATGGCAACAATATAAATAACGTTATTT AAAAAAAGATTGTT
BS I BS II BS III BS IV
-317 transcription start site
-320-330 -310 -300 -290 -280 -270 -260 -250-340-350
Cell Free Extract
A+
G
-Cell Free Extract
A+
G
-6 7 8 9 10
(A)
(B)
Y. Ohtsubo et al. Fig.7
-317-320
-330
-340
-250
-310
-300
-290
-280
-270
-260
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1kb
Y. Ohtsubo et al. Fig. 8
-1284 -400 -242 +3Kmr
-242 +3-1284 -400bphS
pE promoterbphEtnpBPH
terminator
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ATGCATATAATCTATTTTATATAAAAATATAAAAATAACGTTATTTAAAATC
ATGTTCAAAAAAAGATTGTTCGCGCA
TTAGAAAAATGTCGTTTTTTTTCTAAAAAATCAATGGCAACAATATGTTCAA
Tn4371
BS I
BS II
BS III
BS IV
Y. Ohtsubo et al. Fig. 9 by guest on April 17, 2019
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KKS102 +
-+
-+
-+
-
68.8
18.17.7
10.47.6
8.475.5
88.3
∆pE
∆pE∆bphS
∆bphS
BphD activityUnit / mg protein
BphD activity in starin deleted of pE promoter
addition ofstrains biphenyl
BphD activity was measured after 3 hours of incubation with biphenyl
Y. Ohtsubo. et al. table 1
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and Yuji NagataYoshiyuki Ohtsubo, Mina Delawary, Kazuhide Kimbara, Masamich Takagi, Akinori Ohta
degradation in pseudomonas sp. KKS102BphS, a key transcriptional regulator of bph genes involved in PCB/biphenyl
published online July 17, 2001J. Biol. Chem.
10.1074/jbc.M100302200Access the most updated version of this article at doi:
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