bphs, a key transcriptional regulator of bph genes ... · dienoic acid, an intermediate compound in...

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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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L. D. (1998) J Biol Chem 273(36), 22943-9

16. Maxam, A. M., and Gilbert, W. (1977) Proc Natl Acad Sci U S A 74(2),

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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

by guest on April 17, 2019 http://www.jbc.org/ Downloaded from

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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


-317


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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)



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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


-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

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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:

Alerts:

When a correction for this article is posted•

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bphs, a key transcriptional regulator of bph genes ... · dienoic acid, an intermediate compound in...

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