From the Department of Life Science, Sogang
University, Seoul 121-742, Korea and the § School of
Biological Sciences, Institute of Microbiology, Seoul National
University, Seoul 151-742, Korea
Received for publication, November 18, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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Nickel-responsive transcriptional
repression of sodF, which codes for iron- and
zinc-containing superoxide dismutase of Streptomyces griseus, was mediated through an operator ( Aerobic organisms have acquired a specific mechanism to protect
themselves from reactive oxygen species
(ROS)1 such as superoxide
radical (O The Ni-SOD and FeZn-SOD are found as homotetramers of approximately 13- and 22-kDa subunits, respectively, in S. griseus (7) and
S. coelicolor (8). The expression of sodN coding
for Ni-SOD of S. coelicolor (9) and S. griseus2 increased in
the presence of nickel, whereas their FeZn-SOD (sodF) expressions were regulated through the repression of sodF
transcription in the presence of nickel (10, 11). Previously, we found
that the nickel-responsive transcriptional repression of
sodF of S. griseus was exerted through an
inverted-repeat sequence (TTGCAN7TGCAA) overlapping the 5' end (+1, G) of sodF transcript
(11). In addition, nickel-dependent interaction between
cell extracts and the sodF operator DNA was confirmed by
gel-mobility shift assay (11), suggesting the presence of trans-acting
regulatory protein(s) responsible for the repression. To the best of
our knowledge, a nickel-responsive transcriptional regulator has not been described in Gram-positive bacteria, whereas a transcriptional regulator, NikR, has been found for the repression of nik
operon coding for ATP-dependent nickel transport system of
Escherichia coli when nickel is provided in excess (12). An
inverted-repeat sequence in the promoter of nik operon was
proposed as a potential NikR-binding site (13, 14). No sequence
homology was found between the inverted-repeat sequence of E. coli nik operon and the operator of S. griseus
sodF gene.
In this study, we identified srnRQ coding for two small
proteins mediating nickel-responsive transcriptional repression of S. griseus sodF (srn stands for sodF
repression by nickel). They are located
immediately downstream from sodF. SrnR and SrnQ appear to
form an octameric complex composed of four subunits of each protein and
then bind to the sodF operator in a nickel-responsive manner. We propose that DNA binding motif-bearing SrnR is a repressor, whereas nickel-binding SrnQ is a co-repressor. A hypothetical model for
the repression of sodF transcription by the
SrnR·SrnQ complex is presented.
Bacterial Strains and Growth Conditions--
S.
griseus ATCC 23345 was grown at 28 °C in YEME or YMPD
medium as described previously (15, 16). S. griseus
protoplast was prepared as described previously (15, 17) and used for transformation with plasmid DNA, which was isolated from E. coli SURE strain (Stratagene) (18). Transformants were
selected on R2YE agar plate (15). Ampicillin, streptomycin (Sm),
spectinomycin (Sp), kanamycin (Km), and apramycin (Apr) (final
concentrations of 50, 25, 25, 25, and 50 µg/ml, respectively) were
used when E. coli carried the
antibiotic-resistant genes. Km, Apr, and thiostrepton were used
at 5, 5, and 20 µg/ml, respectively, when appropriate for
S. griseus culture.
Nucleotide Sequence Accession Number--
Nucleotide sequence of
0.86-kb SmaI/SalI DNA (Fig. 1), which includes
srnRQ of S. griseus, has been assigned to
GenBankTM under the accession number AF1418663.
DNA Mutagenesis and Plasmid Construction--
The initiation
codon of SrnR and SrnQ were mutated by PCR including overlap extension
as described previously (19). The 1.0-kb
PstI/SalI DNA fragment containing
srnRQ (Fig. 1) was cloned into pBS (Stratagene) to generate
pBSRQ and used as a DNA template. The primer
5'-TGCACCCTTGGAATCAC-3' (A of SrnR ATG codon was replaced by T) was used for the disruption of initiation codon of
SrnR, whereas the SrnQ initiation codon was mutated using the primer 5'-CCTCATAGTCAGGCCT-3' (AG was substituted for
GA corresponding to the third (G) and first base (A) of the first and
second codons of SrnQ, respectively, leaving the SrnR stop codon,
originally TGA, as TAG). The PCR products were
confirmed for the mutations through the DNA sequence analyses followed
by digestion with PstI and SalI and cloning into
PstI/SalI sites of pFD666 (20) to generate
pFDR
Plasmid pIJK365 derived from a low copy number plasmid pXE4 (21) has
the sodF regulatory DNA from Detection of Catechol Dioxygenase Activity--
S.
griseus containing xylE fusion plasmids were cultured
for 48 h on an YEME agar plate. Cells were broken by sonication, and catechol dioxygenase activity was measured with cell extracts as
described previously (21).
Detection of SOD Activity, Preparation of SOD, and Western
Immunoblot Analysis--
Activity staining of SOD in a native
polyacrylamide (10%) gel was performed as described previously (23).
The FeZn-SOD of S. griseus was purified and used as an
immunogen to raise an antiserum from mouse as described previously (7).
The preparation of cell extracts, electrophoresis (SDS-PAGE, 15%
polyacrylamide), and electroblotting of proteins were also performed
according to the methods as described previously (7, 24). A blot
treated with FeZn-SOD-specific antibody was reacted with a 1/5000
dilution of goat anti-mouse IgG (Pierce) conjugated with horseradish
peroxidase, which was visualized using an ECL detection system supplied
by Amersham Biosciences (11). Protein was determined by a modified Lowry method using bovine serum albumin (Sigma) as a standard (25). The
relative SOD activities and FeZnSOD protein levels between samples were
quantified by scanning the gel and blot with Tina 2.0 program of a
BIO-Imaging analyzer (Fuji).
Expression of SrnR and SrnQ in E. coli--
pBSRQ has an insert
of the 1.0-kb PstI/SalI DNA containing
srnRQ (Fig. 1) in the same orientation as lac
promoter of pBS. pBSR Gene Disruption--
A partially digested BamHI
fragment of 4.2 kb containing sodF and srnRQ
(Fig. 7) was cloned into pKC1139 (Aprr) (27), which has a
temperature-sensitive replication origin to generate pKCR. The
EcoN1 site within srnR of the plasmid was interrupted through ligation with 2.2-kb transcription-translation stop
RNA Isolation, Quantification, and Northern (RNA) Hybridization
Analysis--
Total RNA was extracted from S. griseus as
described previously (29). RNA quantification, electrophoretic
separation of denatured RNA, blot transfer, labeling of strand-specific
RNA probe using [ Overexpression and Purification of SrnR and SrnQ Using Fusion to
Glutathione S-Transferase (GST)--
To clone srnR and
srnQ into GST fusion plasmid pGEX-5X-3 (Amersham
Biosciences), initiation and stop codons of the genes were modified to
have BamHI and XhoI sites, respectively. An
N-terminal primer,
5'-AACATGCACGGATCCAATCACGCGCC-3'
(mutated sequence underlined unless otherwise noted),
with the SrnR start codon changed into BamHI site (in
boldface) was used in PCR with a C-terminal primer,
5'-GAGGCCTGATCTCGAGGCGTCGCC-3', containing XhoI site (in boldface) mutated from the SrnR
stop codon. For srnQ, an N-terminal primer,
5'-GGCGACGCCGGATCCTCAGGCCTCG-3' (BamHI site in boldface), and a C-terminal
primer,
5'-CGCGCGGCCGTCTCGAGCCGGGTAGATC-3' (XhoI site in boldface), were used. pBSRQ was
used as a template. The PCR products were digested with
BamHI and XhoI and cloned into the expression
vector pGEX-5X-3 to generate pGEXR and pGEXQ for the expression of SrnR
and SrnQ, respectively. Sequence analyses confirmed the in-frame
insertion into the plasmid as well as no other mutation in the
amplified DNA.
When E. coli DH5 Gel-mobility Shift Assay--
The DNA fragments to be run for
the assay were labeled with [ Far-Western Immunoblot Analysis--
Interaction between SrnR
and SrnQ was analyzed as described previously (32). Native or
SDS-heat-denatured (95 °C for 5 min in 0.2% SDS) SrnR (2 and 5 pmol) was prepared in TDGT buffer (50 mM Tris-HCl, pH 7.9, 0.15 mM NaCl, 1 mM dithiothreitol, 5%
glycerol, and 0.05% Tween 20) with or without nickel followed by
spotting onto nitrocellulose membrane. The nickel was used twice as
much as the protein concentration. The blots were dried at room
temperature for 1 h. After soaking in phosphate-buffered saline
(24) containing 5% nonfat milk at room temperature for 30 min, blots
were incubated in the same buffer containing native SrnQ (15 nM) for 1 h. After washing with phosphate-buffered
saline three times, the membranes were treated with SrnQ-specific mouse
antibodies in the same buffer, reacted with goat anti-mouse IgG
conjugated with horseradish peroxidase, and detected with an ECL
detection system (Amersham Biosciences).
Analytical Gel-filtration Chromatography--
The complex
of SrnR·SrnQ was identified through analytical Sephadex G-100
(Sigma) column (1.2 × 20.0, inner diameter × height (in
cm)). The column buffer was 50 mM Tris-HCl, pH 7.5, containing 100 mM KCl (33). SrnR and SrnQ (60 µg each)
were incubated at 25 °C for the protein interaction in the binding
reaction buffer, which is the same as that used in the gel-mobility
shift assay. The incubation continued with or without nickel (20 µM) for 10 min, and the samples were subjected to the
column chromatography, which was run at room temperature. The
A280 of the eluted fractions was measured, and
SrnR and SrnQ in elutes were quantified using Tricine-SDS-PAGE
(26).
CD Spectroscopy--
Either SrnR or SrnQ (15 µM)
was incubated at 25 °C in 10 mM Hepes, pH 7.6, containing 100 mM NaCl (14) with or without nickel (20 µM). The CD spectra of the proteins were obtained in
wavelength range between 200 and 250 nm at 0.1-nm intervals with J-720
CD spectropolarimeter (Jasco). Five spectra with a resolution of 1 nm,
a scan speed of 50 nm/min, and a response time of 1 s were averaged. The mean residue ellipticity was calculated using the molecular mass of each protein.
Nickel Binding Assay--
Nickel binding of SrnQ and SrnR was
assayed by atomic absorption spectrophotometry following equilibrium
dialysis. 2-ml solution of SrnQ or SrnR (6.4 µM) was
dialyzed against 0.5-liter buffer (10 mM Hepes, pH 7.6, 100 mM NaCl) (14) containing different amounts of
NiCl2 at 4 °C for 48 h. Unbound nickel was then
removed by dialysis in ~500-fold volume of the 10 mM
Hepes, pH 7.6, at 4 °C for 6 h. Metal content was determined
with an AA-880 mark II atomic absorption (flame) spectrophotometer from
Thermo Jarrel Ash. Each reading reported by the instrument was an
average of three determinations. Bovine serum albumin was used as a control.
Nickel-dependent Expression of S. griseus FeZn-SOD Was
Severely Repressed by the Multicopy Presence of the 1.0-kb DNA
Downstream from sodF--
The activity and protein level of FeZn-SOD
were found much more repressed by nickel when the 1.0-kb
PstI/SalI DNA (Fig.
1) downstream from sodF was
maintained in multiple copies in cells (Fig. 2,
pFDRQ). S. griseus
harboring pFDRQ containing the
sodF downstream DNA was grown on YEME agar
plates supplemented with nickel up to 5 µM, and the
FeZn-SOD activity and the protein level were measured at 48 h
after inoculation. Although the FeZn-SOD expression of S. griseus having vector DNA, pFD666, decreased in proportion to
nickel concentration (Fig. 2, pFD666), the presence of pFDRQ
in repeated analyses always lowered the enzyme expression to a greater
extent to display the activity (Fig. 2A) and the protein
level (Fig. 2B) that are barely detectable at 5 µM nickel.
The sodF Downstream DNA Contains Two ORFs, SrnR and SrnQ--
A
sequence analysis of the sodF downstream DNA revealed two
ORFs, which are oriented in the same direction as sodF, and
started at 160 and 501 bp downstream from the SodF stop codon,
respectively (Fig. 1). The first ORF (SrnR) consisted of 114 deduced
amino acids with molecular mass of 12,343 Da, whereas the second
ORF (SrnQ) revealed 110 amino acids of 12,486 Da. The initiation codon of SrnQ overlaps the stop codon of SrnR. A comparison of amino acid
sequence between residues 12 and 82 of SrnR with data base revealed its
homology to the transcriptional regulators of ArsR family (Fig.
3): ~61% similarity (33% identity) to
HlyU, which up-regulates the transcription of hemolysin gene of
Vibrio cholerae (34); 60% similarity (36% identity) to
ArsR as a transcriptional repressor of the arsenical resistance genes
of Staphylococcus xylosus (35); 59% similarity (36%
identity) to the NolR involved in negative control of the expression of
nodulation genes of Rhizobium meliloti (36); 57% similarity
(40% identity) to MerR acting as an activator-repressor protein
involved in mercury resistance of Streptomyces lividans
(37); and 52% similarity (42% identity) to a putative ArsR found in
genome data base of S. coelicolor M145 at Sanger center
(www.sanger.ac.uk/Projects/S_coelicolor/). Around the middle of SrnR
was found a putative helix-turn-helix motif as depicted in Fig. 3. No
protein displaying homology to SrnQ was found. SrnQ has remarkably high
content (26%) of arginine.
Both SrnR and SrnQ Are Required for the Repression of FeZn-SOD
Expression in the Presence of Nickel--
To determine which ORF(s) of
the sodF downstream DNA mediates the enhanced repression of
FeZn-SOD in the presence of nickel, SrnR and SrnQ were mutagenized
through PCR. As illustrated in pFDR
The FeZn-SOD expression of S. griseus harboring either
pFDR SrnR and SrnQ Are Expressed in E. coli--
It was examined
whether srnR and srnQ code for the proteins of
deduced sizes. The proteins were expressed through IPTG induction from
E. coli harboring pBSRQ, pBSR Multicopy Effect of srnR and srnQ on FeZn-SOD Expression Is Exerted
at the Transcriptional Stage through the sodF Operator--
It was
determined whether the effects of srnR and srnQ
in multicopies shown above was exerted at the level of sodF
transcription, and if so, whether the sodF operator was
involved. The sodF::xylE fusion
plasmids, pIJK365 (the wild-type operator) (Fig.
5) and pIMin(+8+9) (the mutated operator)
(Fig. 5), were derived from pXE4, so that they can be compatible with
pFD666 or its derivative, pFDRQ. The introduction of pFDRQ into cells,
which had harbored pIJK365 (Fig. 5, left panel), reduced
~72% XylE activity in the presence of nickel (5 µM)
compared with that without nickel addition, whereas the vector DNA
pFD666 (Fig. 5, left panel) resulted in only 14% decrease
of activity in the same comparison. The result suggested that the
nickel-responsive repression by srnR and srnQ in
multicopies is regulated at the level of sodF transcription. Even without nickel addition, pFDRQ lowered activity (Fig. 5, left panel, Nickel 0), which was interpreted as being due to
the effect of overexpressed SrnR and SrnQ interacting with the residual nickel in YEME medium. On the other hand, pIM5in(+8+9) did not show any
nickel-responsive repression by the presence of pFDRQ (Fig. 5,
right panel). The results indicated that the enhanced repression by srnRQ in multicopies should be exerted through
the sodF operator.
The nickel-dependent repression by pFDRQ was further
confirmed by analyzing the transcription of chromosomal
sodF. S. griseus containing pFDRQ,
pFDR Chromosomal Interruption of srnR Resulted in Derepressed
Transcription of sodF Even in the Presence of Nickel--
The
chromosomal copy of srnR was interrupted with
transcription-translation stop Nickel-dependent Binding of SrnR and SrnQ to the sodF
Operator Was Observed Only When Both Proteins Were Provided--
SrnR
and SrnQ were overexpressed as GST fusion proteins and purified from
E. coli as described under "Experimental Procedures." The purified SrnR and SrnQ showed a molecular mass of ~12 kDa that
were identified by SDS-PAGE (data not shown). The 113-bp StyI-BalI (Fig. 5) DNA containing either
wild-type sequence (Fig. 8A)
or the mutated operator (Fig. 8B, +5 insertion)
was used for gel-mobility shift assay. The wild-type DNA was retarded
only when SrnR and SrnQ were provided together in the nickel-containing reaction mixture. The retarded band was intensified as nickel concentration increased (Fig. 8A). However, neither SrnR nor
SrnQ alone shifted the DNA, even in the presence of nickel (Fig.
8A). On the other hand, the DNA including the mutated
operator did not show any retardation (Fig. 8B), being
consistent with the XylE activities of pIM5in(+8+9) shown in Fig. 5.
Thus, SrnR and SrnQ appear to exert the repressive effect in a
nickel-responsive way through binding to the operator. Because SrnR has
a DNA binding motif, it is expected that SrnR may directly interact
with the operator. However, SrnQ should be present together for the
repression.
The gel-mobility shift of the operator DNA was examined with the two
proteins in different ratios. No retardation was consistently observed
in the absence of one of the two proteins (Fig.
9, lanes 1 and 7).
The amount of the binding complex reached the maximum level when SrnR
and SrnQ were provided simultaneously at 10 µM each
(lane 4). The signal intensity of the binding complex
(C) was ~2-fold higher than that of lanes 3 and
5 in which one of the two proteins was maintained at
half-concentration (5 µM). Thus, the maximum interaction
of SrnR and SrnQ to the sodF operator appears to require
both proteins in 1:1 ratio.
SrnR and SrnQ Directly Interact with Each Other, and the
Interaction Does Not Require Nickel--
Both native (Fig.
10A, spots 1 and
2) and SDS-heat-denatured SrnR (Fig. 10B,
spots 1 and 2) were spotted on nitrocellulose
membrane, and the blots were incubated with native SrnQ followed by
treatment with anti-SrnQ mouse antiserum, which was then detected using anti-mouse IgG conjugated with horseradish peroxidase. The two proteins
in native forms interact with each other by direct binding, even in the
absence of nickel (Fig. 10A, Nickel SrnR and SrnQ Constitute a Hetero-octameric Complex Composed of
Each Protein in 1:1 Ratio--
The formation of SrnR·SrnQ complex
was confirmed through gel-filtration chromatography of Sephadex G-100,
and its molecular weight was measured using the mass calibration curve
drawn with the standard proteins (Fig.
11A). The chromatographic
resolution of either SrnR or SrnQ alone showed a peak, which was
estimated around 10-12 kDa (Fig. 11B, c and
d). Nickel treatment to each protein prior to gel loading
showed the same results (data not shown), which implied that SrnR or
SrnQ by itself, does not form homomultimeric complex. The two proteins
were incubated with or without nickel and then subjected to the column
chromatography. The elution profiles revealed an identical peak
corresponding to ~95 kDa regardless of the nickel treatment (Fig.
11B, a and b). The Tricine-SDS-PAGE of
the 95-kDa fractions showed the two protein bands with equal intensity,
which co-migrated with SrnR and SrnQ (Fig. 11C). Thus, SrnR
and SrnQ appear to form an octameric complex composed of four subunits
of each protein.
Nickel Binds to SrnQ--
The CD spectra of SrnQ were largely
changed after nickel treatment (Fig.
12A, left panel). However,
the spectral patterns of SrnR did not show any significant difference
with respect to nickel (Fig. 12A, right panel). The spectral
change of SrnQ by nickel disappeared following incubation of the
protein mixture with EDTA (50 mM) for 3 h at room
temperature (data not shown). The results suggested that the nickel
binding to SrnQ causes the conformational change of the protein.
Atomic absorption spectrophotometry of SrnQ following equilibrium
dialysis against nickel showed the metal-binding capacity of the
protein that saturates at ~0.80 atom of nickel/molecule with an
apparent Kd of 0.65 µM. Thus, the
stoichiometric balance of at least 1:1 is expected for Ni2+
ion bound to SrnQ. Other metals such as Zn2+ and
Cd2+ were found in the ratio <0.04 metal atom/protein
molecule, and no binding signal was detected with other metal ions
including Ca2+, Mg2+, Cu2+,
Mn2+, and Co2+ (data not shown). In addition,
SrnR did not reveal any metal-binding capacity (data not shown). Thus,
the nickel specifically binds to SrnQ, which appears to be in a
stoichiometric balance of 1:1 (Ni2+/SrnQ).
A highly aerobic organism, S. griseus contains Ni-SOD
and FeZn-SOD for protection against oxidative damages by superoxide radicals. Ni-SOD activity increased after nickel
treatment,2 whereas FeZn-SOD expression is repressed by
nickel at the stage of transcription (10, 11). The transcriptional
repression was exerted through an operator of sodF (11). The
antagonistic production of the two SODs has been proposed as a
regulatory circuit to keep the total SOD activity constant (10). In
this work, we found two ORFs, SrnR and SrnQ, which are responsible for
the nickel-responsive transcriptional repression of S. griseus
sodF. When SrnR and SrnQ were maintained in multicopies in
S. griseus, the Ni-SOD activity was not changed much,
indicating that sodN expression is regulated differently
from that of sodF.
The calculated pI of SrnQ, fairly rich (26%) in arginine, is 12.8, whereas that of SrnR is 7.0. The physiological implication of the basic
pI of SrnQ is not known. The constitutive expression of
srnRQ independent of the presence of nickel was identified using srnR::xylE transcriptional fusion
construct.2 This result explains the observation that the
binding complex with the sodF operator DNA was still
detected in gel-mobility shift assay when nickel was added to the cell
extracts prepared from a nickel-deficient culture of S. griseus.2
The gel-mobility shift band (Fig. 8) was compared with those observed
with the cell extracts of S. griseus. The retarded band with
the purified proteins exactly co-migrated with one (faster-moving band)
of the two binding complexes with the cell extracts (11). The
slower-moving band was not detected, even with up to five times more
the amount of the purified proteins shown in Fig. 8,2 so
the band might reflect additional binding of other proteins from cell
extracts. The probe DNA contains the sodF promoter as well.
The results shown in this work clearly suggest that SrnR interacts with
SrnQ to form an octameric complex, which appears to be composed of four
subunits of each protein (Fig. 13). The
overlapping stop-start codons of SrnR and SrnQ may provide a balanced
translation to maintain 1:1 stoichiometry of the two proteins. The
protein interaction does not require nickel. Our current data explain that SrnQ of the complex is probably a co-repressor of SrnR, because it
can hold nickel at a ratio of one Ni2+/polypeptide and the
ligand binding appears to change the conformation of SrnQ, which may in
turn enhance the DNA binding activity of SrnR through the
protein-protein interaction. The complex then binds to the
sodF operator for transcriptional repression (Fig. 13). A
similar observation has been reported for the interaction between BarA
repressor and BarX co-repressor of Streptomyces virginiae (38-40). They are needed together for binding to the operator site of
barB-varS operon, which codes for a putative
DNA-binding protein and virginiamycin S-specific transport
protein, respectively. BarX was suggested to induce DNA binding ability
of BarA by direct interaction.
2 to +15) spanning over the 5' end (+1) of the transcript. Two open reading frames, SrnR
(12,343 Da) and SrnQ (12,486 Da), with overlapping stop-start codons
were identified downstream from sodF and found responsible for the repression of sodF. The deduced amino acid sequence
of SrnR revealed a DNA binding motif and showed homology to the
transcriptional regulators of ArsR family, whereas SrnQ did not show
any similarity to any known proteins. When srnRQ DNA was
maintained in trans in S. griseus
on a multicopy plasmid, sodF transcription was highly repressed by nickel, but neither srnR nor srnQ
alone showed the effect. Consistently, the sodF
transcription of srnR-interrupted mutant was no longer
repressed by nickel, which was complemented only with srnRQ
DNA. Nickel-dependent binding of SrnR and SrnQ to the
sodF operator DNA was observed only when the two proteins were provided together. The maximum protein-DNA interaction was shown
when SrnR and SrnQ were present in one-to-one stoichiometric ratio. The
two proteins appear to constitute an octamer composed of four subunits
of each protein. SrnR directly interacted with SrnQ, and the protein
interaction did not require nickel. The conformation of SrnQ was
changed upon nickel binding, which was in the ratio of one
Ni2+ ion per protein molecule. A model is proposed in which
SrnQ of the protein complex senses nickel and subsequently enhances the DNA binding activity of SrnR through the protein-protein interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Q and pFDRQ
carrying the mutations in
srnR and srnQ, respectively. Plasmid pFDRQ, also
a derivative of pFD666, contained the same 1.0-kb PstI/SalI DNA including the wild-type
srnRQ (Fig. 1).
365 to +59 (+1, 5' end of
sodF transcript), which was transcriptionally fused to
promoterless xylE DNA (11). pIM5in(+8+9) carries the same
sodF DNA as in pIJK365 with the exception that a 5-bp
nonspecific DNA was inserted between the left and right halves
of the dyad symmetry (11). All plasmids contain
transcription-translation stop
DNA
(Smr/Spr) (22) upstream from the
sodF DNA to block any fortuitous transcriptional read-through originated from the vector DNA.
Q and pBSRQ
harbored
the same insert DNA as in pBSRQ with the exception of mutations in
initiation codon of SrnR and SrnQ, respectively. Plasmids pBSRQ,
pBSR
Q, and pBSRQ
were transformed into
E. coli DH5
. Expression of SrnR and SrnQ was induced by
IPTG at 0.1 mM for 2 h after
A600 of E. coli culture reached
0.6-0.7. SrnR and SrnQ were separated using Tricine-SDS-PAGE (26). The
separating gel of 16.5% T, 6% C was overlaid with a 4% T, 3% C
stacking gel (T denotes the total percentage concentration of both
acrylamide and bisacrylamide, and C is the percentage concentration of
bisacrylamide relative to the total concentration).
DNA (Kmr) (28) following end flushing with Klenow
fragment. The resulting plasmid pKCRKm was introduced into S. griseus, and transformants were selected on YMPD agar plate
containing Km and Apr at 28 °C. Single cross-over recombinants
(Kmr and Aprr) were obtained after incubation
of the transformant at 37 °C. The spores of single cross-over then
were grown in YMPD broth without Apr for 72 h and plated on to the
same medium containing Km. Kanamycin-resistant colonies thus obtained
were examined for their sensitivity to Apr, and finally, three colonies
showing Kmr and Aprs, which were regarded as
srnR-interrupted mutants, were selected among approximately
50 colonies. All of the mutants were confirmed for their chromosomal
arrangements of disruptions by Southern hybridization with probes
labeled with [
-32P]dCTP using DNA-labeling beads
(Amersham Biosciences), and one srnR disruptant, R45,
was chosen for further analyses.
-32P]CTP, and hybridization with the
probe were performed as described previously (11, 30). Transcript
levels were quantified by densitometer scan of the resulting
autoradiograms with the BIO-Imaging analyzer.
containing either pGEXR or pGEXQ grew up
to A600 between 0.6 and 0.7, IPTG was added at
0.1 mM for overexpression of GST-SrnR and GST-SrnQ
proteins. The cells were harvested at 2 h after IPTG induction,
disrupted by sonication, and centrifuged at 4 °C to obtain cell-free
extracts. The GST fusion proteins of ~39 kDa were identified by
SDS-PAGE (12% polyacrylamide). The fusion proteins were purified from
the cell lysate using glutathione-Sepharose 4B (Amersham Biosciences)
followed by elution and concentration of native SrnR and SrnQ after the
removal of GST by Factor Xa (New England Biolabs) digestion. The
purified proteins were assessed for the purity and size by SDS-PAGE
(15% polyacrylamide) (data not shown). The purified SrnR and SrnQ were
used as immunogens to raise antisera as described previously (7).
-32P]ATP using T4
polynucleotide kinase (Promega). The DNA probes (~104
cpm) were incubated at 25 °C with the varying amounts of the purified proteins of SrnR and/or SrnQ in the binding reaction buffer as
described previously (11, 31). After a 10-min incubation with or
without nickel, the reaction mixture was examined using 5%
non-denaturing polyacrylamide gel as described previously (11). The
results provided in this work are typical ones of at least three to
four independent experiments showing virtually the same patterns.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
S. griseus sodF and its
downstream DNA including srnR and
srnQ. The sodF transcription
with respect to nickel is shown. Mutated nucleotides in start and
stop codons of srnRQ are indicated in boldface.
Plasmid pFDRQ contains the 1.0-kb PstI/SalI DNA
fragment containing wild-type srnRQ.
pFDR Q and pFDRQ
are identical to pFDRQ with
the exception of the mutations.
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Fig. 2.
FeZnSOD expression of S. griseus
containing srnR and srnQ in
trans. S. griseus harboring pFD666, pFDRQ,
pFDR Q, or pFDRQ
was grown on YEME agar
plates supplemented with nickel (0, 0.1, or 5 µM) for
48 h. Cell extracts were prepared through sonication.
A, activity staining of SOD was done with 20 µg of
protein/lane in 10% native polyacrylamide gel. B, Western
immunoblot analysis of FeZnSOD was accomplished with 7 µg of
protein/lane.
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Fig. 3.
Sequence comparison of S. griseus
SrnR with bacterial homologues. Deduced amino acid
sequence from residues 12-82 of SrnR was shown, and the sequence
alignment was obtained using the ClustalW, version 1.80, program. Fully
conserved residues are indicated with asterisks, whereas
similar amino acids are marked with dots. The
helix-turn-helix motif is indicated. The size of each protein is
denoted by residue number in parenthesis, and the
residue locations are shown with numbers on each side of the
sequence.
Q (Fig. 1), the
initiation codon of SrnR was changed into TTG resulting in
SrnR
. The SrnR mutation might have polar effect on the
expression of downstream SrnQ, so the 213-bp
XmaIII-EcoNI DNA corresponding to the peptide
from residues 9-78 of SrnR was in-frame deleted to generate pFD(
R)Q
(data not shown). In addition, plasmid pFDRQ
was
constructed to have a substitution of ATA for the initiation codon of
SrnQ, but the SrnR stop codon, originally TGA, was still kept as TAG
(Fig. 1).
Q or pFDRQ
in
trans was compared with that of the control cells containing pFD666 in the presence of nickel (Fig. 2). No significant difference in
FeZn-SOD expression was observed. S. griseus having
pFD(
R)Q showed the same results as those of the cells harboring
pFDR
Q (data not shown), suggesting no polar mutation for
the expression of SrnQ by the SrnR
mutation of
pFDR
Q. Thus, the results clearly indicate that SrnR and
SrnQ are required together for the in trans
effect of the downstream DNA in multicopies.
Q, and
pBSRQ
. The orientation of srnRQ in these
plasmids was the same as that of lac promoter, but their
reading frames were shifted from that of the LacZ
-polypeptide of
the plasmid. The Tricine-SDS-PAGE of the cell extracts of E. coli containing pBSRQ revealed two discrete polypeptides of ~12
kDa only after IPTG induction (Fig. 4,
lane 4). The slow-moving band was regarded as SrnQ, because it was still detected with pBSR
Q (Fig. 4, lane
6). The other fast-moving one was concluded to be SrnR because it
was observed with pBSRQ
(Fig. 4, lane 8). The
sizes of the expressed polypeptides were also similar to the deduced
values of SrnR (12,343 Da) and SrnQ (12,486 Da). We did not believe
that E. coli RNA polymerase(s) recognized the promoter for
srnR and srnQ, because the polypeptides were not
expressed when srnRQ were oriented in opposite direction to
the plasmid lac promoter (data not shown).
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Fig. 4.
SrnR and SrnQ expressed using E. coli lac promoter. E. coli having
pBS (lanes 1 and 2), pBSRQ (lanes 3 and 4), pBSR Q (lanes 5 and
6), or pBSRQ
(lanes 7 and
8) were grown in LB broth at 37 °C until
A600 reached 0.6-0.7 and harvested after IPTG
(0.1 mM) induction for 2 h. Cells were broken, and
total proteins were subject to Tricine-SDS-PAGE. Induced samples were
loaded in lanes 2, 4, 6, and
8, whereas non-induced samples were in lanes 1,
3, 5, and 7. Molecular mass markers
(lane M) are indicated with arrows to the
left of the gel. SrnR and SrnQ were marked with
open and closed arrowheads,
respectively.
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Fig. 5.
Catechol dioxygenase activity of
sodF::xylE transcriptional
fusions containing either wild-type (left panel) and
the mutated operator (right panel) with concurrent
presence of srnR and srnQ in trans
in S. griseus. Plasmid pIJK365
contained the 365-bp upstream DNA from the 5' end of sodF
mRNA. A transcription-translation stop DNA
(Smr/Spr) (22) was cloned at the border between
vector and sodF DNA. The sodF operator of
pIM5in(+8+9) had the insertion of GATCG between +8 and +9. Either pFDRQ
or pFD666 was transformed into cells, which had harbored either pIJK365
(left panel) or pIM5in(+8+9) (right panel), and
transformants were grown on YEME agar plate for 48 h in the
absence (open bar) or presence (shaded bar) of 5 µM nickel. The mean ± S.D. of activities are shown
on each bar.
Q, and pFDRQ
were grown in YEME broth
until exponential phase (A600 = 0.3), and nickel
was added up to 0.1 or 5 µM. Cells were harvested at times indicated and analyzed for the sodF transcript (Fig.
6). The transcript level was largely
decreased at 0.1 µM nickel and barely detectable at 5 µM nickel when pFDRQ was contained in
trans. The reduction of the transcript level by pFDRQ was
also observed without nickel addition, which is consistent with the
lower XylE activity of pIJK365 in the presence of pFDRQ (Fig. 5,
left panel, Nickel 0). The nickel-responsive
repression by pFDR
Q and pFDRQ
was not much
different from that shown by pFD666 (Fig. 6). In addition, the
half-life of sodF transcript of the cells containing pFD666
was ~13 min irrespective of the nickel treatment, which was not
changed at all by the presence of srnR and srnQ
(data not shown). Thus, srnRQ in multicopies highly
repressed the sodF transcription in a
nickel-dependent way.
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Fig. 6.
Northern (RNA) hybridization analysis of
sodF transcript of S. griseus
containing srnR and srnQ in
trans. S. griseus harboring pFD666, pFDRQ,
pFDR Q, or pFDRQ
were grown in YEME broth
until cell growth reached exponential phase
(A600 = 0.3). Total RNA was extracted from the
cells harvested at 30 min (A), 1 h (B), and
6 h (C) after treatment of culture with nickel (0, 0.1, or 5 µM). The sodF transcript (0.8 kb) was
hybridized with RNA probe spanning the gene and its 256-bp upstream DNA
(11).
DNA (Kmr) (28) to
confirm the in vivo effects of overexpressed SrnR and SrnQ.
The interruption at EcoNI site of srnR (Fig.
7A), we believe, also resulted
in a lack of srnQ expression because no XylE activity was
observed with S. griseus containing
srnQ::xylE fusion construct with its
5'-DNA limited to the EcoNI site (data not shown),
illustrating the dependence of srnQ expression on the
regulatory DNA upstream from srnR. The genomic Southern
analysis of srnR-interrupted mutant R45 (Fig. 7A)
revealed the presence of 3.7-kb BamHI bands hybridized to
both srnR (Fig. 7B, left panel) and
the
DNA (Fig. 7B, right panel), confirming
the correct arrangement of chromosomal interruption. Mutant R45 showed
the FeZn-SOD activities and sodF transcription (Fig. 7,
C and D, respectively), which were no longer
repressed by nickel. As expected, the mutation phenotype was
complemented only when pFDRQ (Fig. 2) was introduced into R45 (data not
shown). The results were consistent with the observed effects by
overexpressed SrnR and SrnQ.
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Fig. 7.
Characterization of srnR
disruptant R45. A, chromosomal disruption of
srnR by transcription-translation stop DNA
(Kmr) (28). B, genomic Southern hybridization
analysis of R45 and single cross-over (SCO) recombinant for
comparison with wild type (WT). The chromosomal DNA was
digested with BamHI and probed with PCR-amplified
srnR DNA (left panel) or probed with 2.2-kb
transcription-translation stop
DNA (Kmr) (right
panel). C, the extracts from wild-type and R45 mutant,
which had been grown in the presence of nickel (0, 5, or 20 µM), were used for the activity staining of SOD as
described in Fig. 2. D, Northern (RNA) hybridization
analysis of sodF transcript of wild type and R45. Total RNA
was isolated from the cells harvested at 6 h after treatment of
culture with nickel (0, 5, or 20 µM). The same RNA probe
as used in Fig. 6 was used.
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Fig. 8.
Gel-mobility shift assay of sodF
regulatory DNA (113-bp
StyI/BalI in Fig. 5) with
purified SrnR and SrnQ. Wild-type and mutated sequence of
sodF operator are indicated below the gel. The mutated
StyI/BalI DNA was prepared from pIM5in(+8+9).
Reaction mixtures containing 400 pmol (10 µM) of SrnR
and/or SrnQ were incubated in the absence or presence of nickel (5, 10, and 20 µM). Wild-type DNA (A) or mutated DNA
(B) was used as a probe. Free probe (FP) and
binding complex (C) were indicated with
arrows.
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Fig. 9.
Gel-mobility shift assay of the
sodF regulatory DNA with SrnR and SrnQ in varying
amounts. The wild-type DNA as used in Fig. 8 was incubated with
the proteins in the presence of 20 µM nickel. The
micromolar ratios of SrnR to SrnQ were 0:10 (lane 1), 2:10
(lane 2), 5:10 (lane 3), 10:10 (lane
4), 10:5 (lane 5), 10:2 (lane 6), and 10:0
(lane 7). Free probe (FP) and retarded complex
(C) were indicated with arrows. The relative
levels of C were quantified by densitometer scan of the
resulting autoradiogram.
, spots 1 and 2). Indifference in signal intensity between the
applications of 2 and 5 pmol (spots 1 and 2,
respectively) of SrnR onto the membrane was probably attributed to the
limiting concentration of SrnQ (15 nM) used in binding.
Denatured SrnR did not interact with SrnQ irrespective of nickel
treatment (Fig. 10B, spots 1 and 2). The control immunoblots with SrnQ in native and SDS-heat-denatured forms showed positive signals by anti-SrnQ antiserum as expected (Fig.
10, A and B, spots 3, respectively).
Bovine serum albumin did not show any reaction (Fig. 10, A
and B, BSA), and no cross-reactivity was observed
between SrnR and anti-SrnQ antiserum (data not shown). The analyses
with SrnQ on the membrane followed by sequential reactions with SrnR,
anti-SrnR antiserum, and anti-mouse IgG-horseradish peroxidase
displayed the same results (data not shown). From the results, it was
evident that the interaction between the two proteins does not require
nickel and that the protein-protein interaction can take place before
binding to the operator DNA.
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Fig. 10.
Far-Western immunoblot analysis for the
interaction between SrnR and SrnQ. Native SrnR (panel
A) or the denatured SrnR (95 °C for 5 min in 0.2% SDS)
(panel B) in TDGT buffer (32) were spotted onto
nitrocellulose membrane in the absence (Nickel ) or the
presence (Nickel +) of nickel. The nickel concentration was
twice as much as the protein concentration. Spots 1 contained 2 pmol of SrnR, whereas spots 2 and
3 had 5 pmol of the proteins. The blots were incubated with
native SrnQ (15 nM) followed by reaction with anti-SrnQ
antiserum. Native and denatured SrnQ (panels A and
B, spots 3, respectively) were used as positive
controls, whereas the native and denatured bovine serum albumin
(panels A and B, BSA, respectively)
were included as negative controls. BSA, bovine serum
albumin.
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Fig. 11.
Analysis of SrnR·SrnQ complex using
gel-filtration chromatography. A, the mass calibration
curve for Sephadex G-100 column (1.2 × 20.0, inner diameter × height (in cm)) was drawn with the standard proteins including
aldolase (117 kDa), phosphorylase b (94 kDa), albumin (67 kDa),
ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor
(20.1 kDa), and -lactalbumin (14.4 kDa). B, SrnR and SrnQ
(60 µg each, 15 µM) were incubated without
(a) or with (b) 20 µM nickel for 10 min and were subjected to the chromatography. Each of SrnQ
(c) and SrnR (d) (40 µg each, 10 µM) was loaded as controls. C, the fractions
for peaks were analyzed using Tricine-SDS-PAGE. The fractions (10 µg
each) pooled for the 95-kDa peaks without or with nickel (panel
B, a and b) were included in lanes
1 and 2, respectively, whereas the peak fractions (5 µg each) for SrnQ (Panel B, c) and SrnR
(Panel B, d) were loaded in lanes 3 and 4, respectively. 50 µg of the purified SrnQ
(lane 5) and SrnQ (lane 6) were included as
controls. The molecular mass markers (lane M) are shown to
the left.
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Fig. 12.
Nickel binding to SrnQ. A,
CD spectra of SrnQ (left panel) and SrnR (right
panel) treated with or without nickel (20 µM).
Proteins were used at 15 µM. B, nickel-binding
saturation curve of SrnQ (6.4 µM). Nickel binding was
determined by equilibrium dialysis against varying concentrations of
nickel followed by atomic absorption spectrophotometry. , SrnQ;
,
bovine serum albumin (negative control).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 13.
A model illustrating
nickel-dependent repression of sodF
transcription by the complex of SrnR·SrnQ. The
sodF operator was indicated with facing arrows
(11). Nickel ion was shown as closed circles. R
and Q denote SrnR and SrnQ, respectively.
A nickel-sensing transcriptional repressor NikR of E. coli has been reported as a direct sensor of nickel ion to negatively regulate nikABCDE expression by binding to an operator consisting of two 5'-GTATGA-3' half-sites related by dyad symmetry and separated by 16 base pairs (14). Roughly, a one-nickel ion was proposed to bind to each NikR subunit of the acting dimeric complex (41). No homology between the primary structures of SrnR and NikR was found.
Taken together, the experimental results presented here provide an
interesting example of transcriptional repression by the protein
complex composed of the DNA binding motif-bearing repressor and
nickel-binding co-repressor. The detailed nature of the protein-protein interaction between the repressor and co-repressor remains to be determined.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF1418663.
¶ To whom correspondence should be addressed: Dept. of Life Science, Sogang University, Mapo, Shinsu 1, Seoul 121-742, Korea. Tel.: 82-2-705-8459; Fax: 82-2-704-3601; E-mail: jgklee@ccs.sogang.ac.kr.
Published, JBC Papers in Press, March 17, 2003, DOI 10.1074/jbc.M211740200
2 J.-S. Kim and J. K. Lee, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
ROS, reactive oxygen species;
SOD, superoxide dismutase;
Sm, streptomycin;
Sp, spectinomycin;
Km, kanamycin;
Apr, apramycin;
GST, glutathione
S-transferase;
ORF, open reading frame;
XylE, cathechol
dioxygenase;
IPTG, isopropyl-1-thio--D-galactopyranoside;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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