(Received for publication, December 16, 1996)
From Mutations that exhibit susceptibility to
acriflavine have been isolated and classified as acr
mutations in Escherichia coli. We cloned the
acrB gene, which has been identified as a mutation of the
gyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of
the gyrase B subunit. The mutant B subunit was found to associate with
the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in
the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent
stimulation was observed infrequently. A gel shift assay showed that
acriflavine inhibited the DNA binding of gyrase. The acrB
mutation also reduced significantly the DNA binding of gyrase but did
not change the sensitivity to acriflavine. These results revealed that
the acrB mutation is related to the inhibitory mechanism of
acriflavine; and the acriflavine sensitivity of the mutant, at least
in vitro, is caused mainly by reduction of the enzyme
activity. Further, our findings suggest that the COOH-terminal region
of the B subunit is essential for the initial binding of gyrase to the
substrate DNA.
Acriflavine is an acridine dye that causes inhibition of cell
division in microorganisms, plasmid loss of bacterial cells, and high
mutation frequencies. The mutagenic activity of acridine dyes results
mainly in frameshift mutation, which is derived from insertion or
deletion of base(s) in the DNA (1). Although acriflavine is known as an
antibiotic agent, almost all Escherichia coli K12 wild-type
strains exhibit resistance to this reagent. For investigation of
determinants in acriflavine resistance, five acridine-sensitive mutants
have been isolated: acrA (2), acrB (3),
acrC and acrD (4), and acrE (5).
The acrA mutant is highly cross-sensitive with
membrane-attackable substances such as phenethyl alcohol, detergents,
and some fatty acids (6, 7). The cloned acrA+
gene showed a gene dosage effect in E. coli (8). Sequence analysis revealed an operon encoding two membrane protein genes acrA and acrE (this is not the same gene
described above) (9). This acrAE operon is homologous to the
envCD operon mutant, which is also hypersusceptible to basic
dyes, detergents, and antibiotics (10). These genes are deduced to be
subunits of energy-dependent pump proteins.
Mutations at the acrA locus also occurred in response to
deletion of topA, which encodes topoisomerase I (11). These
mutations help the survival of The DNA gyrase of E. coli consists of two A subunits
(Mr = 105,000, gene gyrA) and two B
subunits (Mr = 95,000, gene gyrB) to
form a tetramer. This enzyme catalyzes the reaction of
supercoiling-relaxation, knotting-unknotting, and
catenation-decatenation for DNA strands in the presence of ATP and
divalent metal ions (14-17). The A subunit contains the active site
for DNA breakage and reunion, and it forms a homodimer in the absence
of the B subunit that does not make a dimer conformation itself (18).
The B subunit has an ATPase domain (NH2-terminal region),
the structure of which has been demonstrated crystallographically (19).
The B subunit that lacks the NH2-terminal ATPase domain,
named Here we report the cloning and characterization of the acrB
mutation located in the COOH-terminal region. The results suggest that
this region contributes to the DNA binding of gyrase.
Wild-type strain W1895
(metB, gyrA+,
gyrB+, acriflaviner) and
acrB strain N2879 (acriflavines mutant derived
from W1895) were used in the acriflavine resistance test (3). The
plasmid pJB11, which has 3.4-kilobase fragment containing the
gyrB gene (22), was kindly provided by Dr. A. Hase (Osaka
City Research Institute of Public Health and Environmental Science).
Expression vector pGEX4T-3 was purchased from Pharmacia Biotech Inc.
pBluescript II (KS+) was from Stratagene.
PGY medium-concentrated (1%
Polypeptone (Difco Laboratories), 0.3% yeast extract, 0.3% NaCl,
0.1% glucose, adjusted pH 7.4) and PGY medium-dilute (0.5%
Polypeptone, 0.1% yeast extract, 0.3% NaCl, 0.1% glucose, adjusted
pH 8.0) were used for the acriflavine sensitivity test. Other media
were constructed as described in Ref. 23. Restriction enzymes were from
Takara Shuzo (Japan), the glutathione S-transferase gene
fusion system was from Pharmacia, and oligonucleotides were from
Biologica (Japan).
Genomic DNA was purified
from E. coli strain N2879 as described by Cosloy and Oishi
(24). The DNA library was constructed with the pBluescript II
(KS+) plasmid, and the gyrB(acrB) gene was
screened by colony hybridization using a wild-type gyrB gene
as a probe. The DNA fragment was labeled with the ECL system (Amersham
Corp.). The gyrA gene was cloned from strain W1895 by
polymerase chain reaction using Vent DNA polymerase (New England
Biolabs). DNA sequencing was performed using Sequenase version 2.0 (U. S. Biochemical Corp.)
gyrA, gyrB, and
gyrB(acrB) gene products were purified by the protocol of
the glutathione S-transferase fusion protein system, DEAE-Sepharose column (25), and a Novobiocin-Sepharose column (26).
Each plasmid for overexpression of the gyrase subunit was constructed
using the expression vector pGEX4T-3 (Pharmacia). In the expression
plasmid for the gyrA gene, an arginine codon AGA at the 6th
codon, which is a rare codon in E. coli, was replaced by
CGT. All of the gyrase genes were inserted in the site of
BamHI (NH2 terminus) and XhoI (COOH
terminus) of the vector; each gene product is expected to have two
extra amino acids upstream of the first methionine after the thrombin
cleavage. The transformed cells were inoculated into 2.5 liters of
2 × YT medium and incubated at 30 °C with vigorous shaking
until A600 reached at 0.5. Expression of the
fusion gene was induced by addition of isopropyl
To purify the B subunit, the eluate from the glutathione-Sepharose
column was dialyzed overnight against 3 liters of TGED buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10% glycerol,
5 mM DTT, 1 mM phenylmethylsulfonyl fluoride)
containing 0.12 M NaCl and loaded onto a DEAE-Sepharose
(type DE52, Whatman) column (12-ml bed volume) previously equilibrated
with the same buffer. The flow-through fraction was collected and
subjected to the Novobiocin column procedure. To obtain the A subunit,
the DEAE column procedure was skipped.
The sample was dialyzed overnight against 3 liters of HEPES buffer (25 mM HEPES, pH 8.0, 50 mM KCl, 1 mM
EDTA, 5 mM DTT, 10% ethylene glycol) and loaded onto a
Novobiocin affinity column (50-ml bed volume) equilibrated with the
same buffer. To obtain the A subunit, the flow-through fraction was
collected. For elution, HEPES buffer containing 5 M urea
was used to obtain the wild-type B subunit and HEPES buffer containing
7 M urea for the acrB mutant subunit.
Finally, these samples were dialyzed overnight against 3 liters of TGED
buffer without NaCl, concentrated by Centricon (Amicon), and stored at
Relaxed pBluescript plasmid DNA was
prepared by topoisomerase I enzyme from rat liver (27). Gyrase enzyme
was reconstituted in 30 µl of reconstitution buffer (20 mM Tris-HCl, pH 7.6, 0.2 mM EDTA, 70 mM KCl, 10 mM MgCl2, 5 mM DTT, 360 µg/ml BSA, 20% glycerol) with purified A and
B subunits (7.5 µg each). The supercoiling assay was performed in 60 µl of the reaction mixture containing 50 mM Tris-HCl, pH
7.5, 20 mM KCl, 10 mM MgCl2, 10 mM DTT, 1.5 mM ATP, 5 mM
spermidine, 50 µg/ml BSA, 500 µg/ml tRNA, 10% glycerol, 1 µg of
relaxed pBluescript DNA, and various amounts of reconstituted gyrase.
Samples were incubated at 30 °C for 30 min, then extracted with
phenol solution and chloroform:isoamyl alcohol solution twice, respectively. DNA was recovered by ethanol precipitation, dissolved in
TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), and
subjected to 0.8% agarose gel electrophoresis. One unit of enzyme
activity of gyrase was defined as the amount that brought 50% of
relaxed DNA to the supercoiled position in agarose gel
electrophoresis.
The reaction mixture (100 µl) contained 50 mM Tris-HCl, pH 7.5, 20 mM KCl, 10 mM MgCl2, 50 µg/ml BSA, 1 µg of gyrase, and various concentrations of ATP with or without 2.5 µg of relaxed pBluescript II (KS+) DNA. The mixtures were incubated at
30 °C for 10 min. The amount of generated ADP was measured by high
performance liquid chromatography as described by Taylor et
al. (28).
The gyrase cleavage site of pBR322 (29)
(base position 886-1089) was amplified by polymerase chain reaction
and subcloned into pBluescript. The isolated DNA fragment was labeled
with [ Using
the gyrB(wild) gene as a probe, we isolated the
gyrB(acrB) gene from N2879 strain. The nucleotide sequences
of the gyrB(acrB) gene revealed two nucleotide
substitutions. The 2275th A from the translation start was substituted
for C by transversion, and 2278th C was substituted for T by
transition. These point mutations alter amino acids in the B subunit:
Ser759
The bacterial growth curves in the presence of 20 µg/ml
acriflavine are shown in Fig. 2. Wild-type strain
(W1895) showed continuous growth without time lag. The wild-type cells
carrying a high copy number of plasmids with wild-type gyrB
(pJB11) or gyrB(acrB) (pSB1) had resistant features.
However, the cells carrying pJB11 showed more intense growth than the
cells without plasmid, whereas the cells carrying pSB1 showed a 2-h
time lag for growth recovery. In the mutant strain (N2879), the cells
without plasmid and with pSB1 did not show growth. On the contrary,
mutant cells carrying pJB11 showed recovery of the survival after a 4-h
decline. Such recovery after a limited time may result from the time
lag for gyrB expression induced by decreased superhelicity
of the genome DNA (32). The hypersensitivity to acriflavine in the
N2879 strain has a complex feature that cannot be explained by the gene
dosage effect alone. However, the semidominant character of the
gyrB(acrB) gene, which would be caused by the stoichiometric
effect of the gene product, is notable.
To analyze the gene product
of acrB, each subunit of gyrase was overexpressed and
purified using the glutathione S-transferase-fusion protocol. After the glutathione S-transferase column
procedure, however, a small amount of the other partner of the subunit
was still found in purification of both A and B subunits, and each sample exhibited supercoiling activity. Therefore, these glutathione S-transferase-fusion proteins seem to retain the ability to
associate with the partner subunit even though they contain a large
polypeptide of glutathione S-transferase in the
NH2-terminal side. The copurified A and B subunits were
absorbed and eliminated by a DEAE-Sepharose column and a Novobiocin
affinity column, respectively. Absence of the partner subunit was
confirmed by SDS-polyacrylamide gel electrophoresis and supercoiling
assay. About 10 mg of protein was obtained from a 2.5-liter culture in
each case.
The supercoiling activity in
vitro was detected from gyrase proteins reconstituted with both
wild-type and the mutant B subunit. However, their specific activities
were quite different. The specific activity of wild-type gyrase was
calculated to be 3.2 × 106 units/mg of protein, and
that of mutant gyrase was 4.0 × 104 units/mg.
Therefore, the activity of gyrase was reduced 80-fold by
acrB mutation.
We also examined the effect of acriflavine in the supercoiling reaction
(Fig. 3). Acriflavine caused partial inhibition against 1 unit of gyrase at a concentration of 1 µg/ml and complete
inhibition at 6 µg/ml. Against 100 units of gyrase, little inhibition
was observed at 10 µg/ml. These findings were common to the wild-type and mutant gyrase. Thus, the sensitivity of the supercoiling activity to acriflavine was not affected by the mutation.
We examined whether the mutant
B subunit had lost the ability to associate with the A subunit. The
molecular weight of the reconstituted mutant gyrase protein was
estimated by a Superdex-200HR (Pharmacia) column, and the eluted
proteins were analyzed by SDS-polyacrylamide gel electrophoresis (Fig.
4). Both wild-type gyrase and mutant gyrase were eluted
at the same position with a molecular mass of 400 kDa, which
corresponds to that of regular gyrase tetramer. SDS-polyacrylamide gel
electrophoresis revealed an equal amount of gyrA and
gyrB subunits. These results showed that the mutant B
subunit was able to construct a gyrase tetramer, indicating that the
lower supercoiling activity of the mutant gyrase was not due to the
loss of the affinity with the A subunit.
The NH2-terminal region of the B
subunit contains an ATPase domain. We examined the ATPase activity of
wild-type and mutant gyrase (Fig. 5). In the absence of
DNA, these enzymes showed a similar level of intrinsic ATPase activity.
The Lineweaver-Burk plot indicated that both Km for
ATP was 1.7 mM as reported (26). On the other hand, in the
presence of DNA, the ATPase activity was quite different in the
wild-type and mutant; the ATP hydrolysis of wild-type gyrase was
significantly stimulated by DNA, whereas that of mutant gyrase was
hardly stimulated.
Fig. 6 shows the inhibitory effect of acriflavine on
ATPase activity. Although slight inhibition was observed under the
conditions without DNA, the inhibitory effect was the most marked in
the case of wild-type enzyme with DNA. In the case of both wild-type and mutant gyrase, the activity was reduced by the high concentration of acriflavine (>3 µg/ml) to the same level as the intrinsic ATPase activity. These results indicated that acriflavine specifically inhibited the DNA-dependent ATPase activity.
We examined DNA binding of
wild-type and mutant gyrases by gel shift assay. The wild-type gyrase
showed notable binding to a 204-base pair DNA fragment containing the
cleavage region of gyrase (29) (Fig. 7A). On
the other hand, the gyrase with gyrB(acrB) subunit showed
very little binding capacity (Fig. 7B). The extent of
gyrase-DNA complex formation decreased as the concentration of
acriflavine increased in the case of wild-type and mutant gyrase. Fifty
percent inhibition of binding was observed at a similar concentration
of the acriflavine (~20 µg/ml) in these experiments, indicating
that the mutation did not change the sensitivity to acriflavine in DNA
binding. Fig. 8 shows the DNA binding of gyrase under
the competitive conditions of B subunits to associate with the A
subunit. Various amounts of wild-type and mutant B subunits were mixed
with constant amount of the A subunit, and the mixtures were subjected
to the gel shift assay. With an increasing amount of the mutant B
subunit, the amount of the gyrase-DNA complex decreased (Fig.
8A). On the other hand, an excess amount of wild-type B
subunit did not inhibit DNA binding and rescued the defect of the
mutant gyrase (Fig. 8B).
Gyrase is a target protein of several antibiotics, some of whose
mechanisms have been analyzed in detail. Coumarin drugs bind the
NH2-terminal domain of the B subunit and inhibit ATP
hydrolysis of the enzyme. Quinolone antibiotics are deduced to bind a
transient complex consisting of cleaved DNA and gyrase, which are bound covalently to each other. Analysis of the resistant mutations of the
gyrase gene revealed that mutations occurred in the corresponding domains where these antibiotics act. Isolated mutant gyrases showed resistant features in vitro as well as in vivo
(33-35).
The present work showed that acriflavine suppressed DNA binding of
gyrase (Fig. 7), and the acrB mutation caused the reduction of DNA binding (Figs. 7 and 8). These results suggest that the acrB mutation is also related to the antibiotic mechanism as
the resistant mutations against coumarin and quinolone. Acriflavine is
well known as a DNA intercalate substance, and it changes the superhelical density of plasmid as ethidium bromide does, implying that
it changes the conformation of the DNA strand. We found that similar
concentrations of acriflavine were effective in inhibiting the
wild-type gyrase and an 80-fold higher amount of the mutant gyrase in
the supercoiling assay (Fig. 3). Therefore, acriflavine is most likely
to interact with DNA and not directly with the gyrase protein. As the
DNA binding is the initial step of gyrase function, it is reasonable to
consider that acriflavine suppresses the subsequent processes such as
ATPase activation (Fig. 6) and DNA supercoiling (Fig. 3).
The acrB mutation, however, did not change the sensitivity
of gyrase to acriflavine in vitro, unlike the resistant
mutations against coumarin and quinolone, which change the sensitivity
of the gyrase to them, respectively. Supercoiling assay and gel shift assay (Figs. 3 and 7) indicated that the mutant enzyme reduced the
activity without becoming more sensitive to acriflavine. These findings
suggest that the acriflavine-sensitive feature of acrB is
mainly the result of a reduction of specific activity. When we prepared
the cell extract from strain N2879 with partial purification by
polymine P precipitation (27), no supercoiling activity was detected in
that extract, whereas the expected activity was found in the extract
from the wild-type strain (data not shown). Thus, gyrase activity
in vivo also seemed to be decreased. The mechanism of the
susceptibility to acriflavine in vivo, however, appears too
complex to be elucidated. For example, the reduction of supercoiling activity may cause a decrease in the superhelical density of genome DNA
which allows acriflavine to have greater access to DNA.
The acrB mutation alters two amino acids (S759R/R760C) near
the COOH-terminal end of the gyrase B subunit (Fig. 1). The region designated as C-TERM in Fig. 1 is separated from the
NH2-terminal part of the B subunit by an insertion of about
a 170-amino acid sequence (black boxed in Fig. 1), which is
absent in gyrases of Gram-positive bacteria, bacterial topoisomerase IV
(parE gene), and eukaryotic topoisomerase II. The C-TERM is
expected to be a separable structural domain, but the relationship to
DNA binding has not been reported in the region. Fig.
9A shows the homology of the C-TERM region to
the corresponding regions of B subunit of Bacillus subtilis
gyrase and parE protein of E. coli topoisomerase IV. As the deletion of 29 amino acids from the COOH-terminal end of
parE protein (double underlined in Fig.
9A) results in a loss of ability to associate with
parC protein (counterpart of the gyrase A subunit) (36),
C-TERM potentially includes a structure necessary for association with
the A subunit. Thus we examined whether the acrB mutation
affects the association with the A subunit. The mutant B subunit,
however, showed normal assembly with the A subunit in the gel
filtration assay (Fig. 4) and the gel shift assay (Fig. 8). These
results suggest that the reduction of DNA binding is caused by an
alteration in the C-TERM region itself, not by loss of the quaternary
structure.
Berger et al. (37) have recently reported the crystal
structure of yeast topoisomerase II and pointed out that a region including a highly conserved sequence (Fig. 9A), which is
located at about 20 residues NH2-terminal from the
acrB site, is involved in the contact with the other B
subunit in the gyrase tetramer. Considered together with the A
subunit-associating region described above, the C-TERM region appeared
to serve the two functional structures that are related to contact with
the partner A and B subunits, respectively. The crystal structure of
topoisomerase II indicates that the counterpart of the region between
the conserved sequence and acrB is composed of an Several possibilities are expected to reduce the DNA binding affinity.
One possibility is that the acrB mutation affects DNA recognition and/or the subsequent conformational change of gyrase which is induced during DNA binding. From the analogy of crystal structure of topoisomerase II, acrB seems to be located near
the active site tyrosine and CAP-like DNA binding domain in the A subunit. The hydrophobicity plot shows that the mutation region is
hydrophilic (Fig. 9B), implying that the amino acid residues are located at the surface of the protein. Further, the acrB
mutation changes the location of a positive charge (Arg760)
which might interact directly with DNA. Therefore, it is possible to
speculate that this region is related directly to the recognition and/or transportation of DNA to the DNA binding domain of the A
subunit. In fact, DNA binding is suggested to cause a structural change
of the region corresponding to the COOH-terminal part of C-TERM (38).
The amino acid sequence near the acrB mutation region is
well conserved in the E. coli parE gene product as well as
in B. subtilis gyrase (Fig. 9A). DNA binding
characteristics of topoisomerase IV are thought to be more similar to
those of type II eukaryotic enzymes than those of the gyrases from the observation that topoisomerase IV protects only short region (34 base
pairs) of DNA from micrococcal nuclease digestion as eukaryotic topoisomerase II (40). Thus, the acrB mutation region seems to be related to general DNA binding and not to the gyrase-specific DNA
wrapping, which is thought to be caused by the 33-kDa domain of the A
subunit (17).
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBankTM/DDBJ Data Bank with accession number(s) D87842[GenBank]. We thank Dr. G. Katsuura and Dr. M. Ishizuka
for helpful advice on the ATPase assay and the gel mobility shift
assay, respectively.
Aburahi Laboratories,
Biological Institute,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
topA strains under
specific growth conditions. Deletion of topA is deleterious
to E. coli, and several compensatory mutations have also
been mapped to gyrA or gyrB (11). Although the
relationship between acrA and the superhelicity of genome DNA has not been elucidated clearly yet, it is interesting that the
acrB gene has been demonstrated to fall into the same
cistron of gyrB (12); in the genetic map of the E. coli chromosome, acrB is designated in the locus of
gyrB (13). These lines of evidence suggest that gyrase is at
least one of the target proteins of acriflavine.
protein, still shows DNA relaxing activity (20, 21).
Therefore, this region is thought to contain all of the functional
domains in the B subunit for establishing topoisomerase activity. The
mechanisms of the precise parts of this protein, however, have not been
elucidated yet.
E. coli Strains and Plasmids
-D-thiogalactopyranoside to 0.1 mM. After 30 min (15 min for B subunits), the culture was cooled down rapidly on
ice, and cells were harvested. Further incubation caused a formation of
an inclusion body that was hardly solubilized. The procedure of the
glutathione-Sepharose column was essentially as described in
manufacturer's protocol (Pharmacia). We used phosphate-buffered saline
containing 10% glycerol, 5 mM DTT1 and 1 mM
phenylmethylsulfonyl fluoride for column washing, thrombin cleavage.
and protein elution.
80 °C.
-32P]ATP by T4 polynucleotide kinase. The
binding reaction was performed with the method of Bachellier et
al. (30) with modifications. Gyrase was reconstructed with 5.0 µg of A and B subunit, respectively, in 20 µl of the reconstitution
buffer described above. Half of the reconstituted gyrase (10 µl) was
added to 10 ml of 2 × binding buffer (80 mM Tris-HCl,
pH 7.6, 12 mM MgCl2, 40 mM KCl, 4 mM DTT, 20 mM ATP) containing 300 pg of labeled
DNA and 25 ng of double stranded competitor DNA
(poly(dI-dC)·poly(dI-dC), Sigma). Subsequently, samples were
incubated at 25 °C for 60 min. Polyacrylamide gel (multigel 2-15%
gradient, Daiichi-kagaku Co. Ltd.) was used, and prerunning was done
with 25 mM Tris-HCl, pH 8.4, 192 mM glycine, 8 mM MgCl2 at 90-V constant voltage for 3 h
at 4 °C. Protein-bound and free DNA were separated
electrophoretically with new buffer at 90-V constant voltage for 4 h at 4 °C. Gels were dried and contacted to the x-ray film. To
quantitate the gyrase-DNA complex, a gel containing the complex was
excised, and the radioactivity was counted by the Cerenkov method.
Identification of the acrB Mutation in the gyrB Gene
Arg and Arg760
Cys. These
mutations were located at the COOH-terminal region of the
gyrB gene (Fig. 1). The nucleotide sequence
of the other part was identical to the reported gyrB
sequence (31).
Fig. 1.
A scheme of the domain structure of E. coli gyrase B subunit and the acrB mutation
site. The ATPase domain boundary is defined by preferred trypsin
cleavage site (Arg393) (41). The black box
region shows the inserted amino acid sequence that is not found in
B. subtilis gyrase and eukaryotic topoisomerase; and the
boundary region is taken from Wyckoff et al. (42). The domain designated by Q contains mutation sites of
quinolone-resistant genes (Asp426, Lys447,
denoted by arrow) and the region of conserved amino acid
motifs which is denoted by the horizontally hatched box:
EGDSA (424-428), PL(R/K)GK(I/L/M)LN (445-452), IM(T/A)D(JQ/A)D
(495-500). The COOH-terminal region neighboring the inserted sequence
designated as C-TERM (see "Discussion"). The mutation sites of
acrB are in the C-TERM region: Ser759 and
Arg760 (this paper). The expected A subunit association
region is indicated by the cross-hatched box (see
"Discussion").
[View Larger Version of this Image (22K GIF file)]
Fig. 2.
The growth of E. coli in the
presence of 20 µg/ml acriflavine. The bacterial cells were
cultured in the PGY medium-concentrated for 18 h at 37 °C and
washed three times with saline solution (0.8% NaCl) by centrifugation.
The cells were diluted in the PGY medium-dilute (105
cells/ml) containing 20 µg/ml acriflavine (AF) and shaken
for various duration. After washing with a saline solution, the cells were plated onto the PGY medium-concentrated to count viable cells. , W1895 (wild-type strain);
, W1895 with pJB11(gyrB);
, W1895 with pSB1(gyrB(acrB));
, N2879
(acrB mutant);
, N2879 with pJB11;
, N2879 with
pSB1.
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
Inhibitory effect of acriflavine on DNA
supercoiling activity. Supercoiling assay was performed with 1, 10, and 100 units of reconstituted gyrase containing wild-type or
mutant B subunit in the presence of various concentrations of
acriflavine (AF). In the experiments of mutant gyrase,
80-fold higher amounts of the enzyme were used for the assays because
of its reduced activity (see "Results"). R and
S represent the positions of relaxed and supercoiled
plasmids, respectively.
[View Larger Version of this Image (41K GIF file)]
Fig. 4.
Estimation of subunit assembling. A
subunit (15 µg) was mixed with 15 µg of wild-type or mutant B
subunit in 100 µl of reconstitution buffer, as described under
"Experimental Procedures," except for glycerol. After incubation
for 30 min at 20 °C, the mixture was applied directly to a gel
filtration column (Superdex-200HR, Pharmacia) which was equilibrated by
reconstitution buffer without BSA and glycerol. The proteins in peak
fractions were collected, concentrated, and analyzed by
SDS-polyacrylamide gel electrophoresis. The protein at the position of
66.2 kDa is BSA in the reconstitution mixture.
[View Larger Version of this Image (26K GIF file)]
Fig. 5.
Intrinsic and DNA-dependent
ATPase activity of wild-type and mutant gyrase. ATPase activity of
wild-type (panel A) and mutant (panel B) gyrase
was determined with various ATP concentrations. , without DNA;
,
with 2.5 µg of pBluescript DNA.
[View Larger Version of this Image (15K GIF file)]
Fig. 6.
Inhibitory effect of acriflavine on ATPase
activity. Reaction mixtures were incubated with 1 µg of gyrase
in the presence of 2 mM ATP and various amounts of
acriflavine (AF). , wild-type gyrase with DNA;
,
wild-type gyrase without DNA;
, mutant gyrase with DNA;
, mutant
gyrase without DNA.
[View Larger Version of this Image (13K GIF file)]
Fig. 7.
Inhibitory effect of acriflavine on DNA
binding. Gel shift assay was performed with wild-type (panel
A) or mutant (panel B) B subunit and the binding buffer
containing various concentrations of acriflavine (AF).
Upper panels show the autoradiography of gel mobility shift
assay. Lower panels show quantitative results from the
radioactivity in DNA-gyrase complexes. Arrows indicate the
positions of the DNA-gyrase complex.
[View Larger Version of this Image (37K GIF file)]
Fig. 8.
Competitive effect of mutant and wild-type B
subunit on DNA binding. A subunit (7.5 µg) was mixed with an
equal amount (7.5 µg) of wild-type B subunit and various amounts of
mutant B subunit (panel A) or mixed with an equal amount of
mutant B subunit and various amounts of wild-type B subunit
(panel B) in the reconstitution buffer. These mixtures were
used for gel shift assays, and the radioactivity in DNA-gyrase complex
was counted.
[View Larger Version of this Image (18K GIF file)]
Fig. 9.
Homology and hydrophobicity plot of mutation
region. Panel A, comparison of the amino acid sequences of
C-TERM region of E. coli gyrase B subunit (gyrB)
and corresponding region of B. subtilis gyrase
(subB) and E. coli parE product
(parE). Superscript and subscript
letters in E. coli gyrase indicate the wild-type and
acrB mutant sequence, respectively. Identical amino acids are indicated by two dots, and conservative differences are
indicated by a single dot. Conserved amino acids in type II
topoisomerase enzymes are indicated by open boxes.
Arrows indicate the amino acid residues expected to contact
to the other B subunit (37). The double underlined sequence
in parE is putative association region with parC
protein (A subunit homolog) (36; see "Discussion"). The numbers of
amino acid residues correspond to the position of E. coli
gyrase. Panel B, hydrophobicity plot of the C-TERM region of
E. coli gyrase. Hydropathy values were computed according to
Kyte and Doolittle (43). The horizontal scale is adjusted to
the amino acid sequence in panel A. The region of
acrB mutation is indicated by a bar.
[View Larger Version of this Image (24K GIF file)]
-helix
and forms an axis that extends to the linker between the A
and B
subfragments. Therefore, the axis-like structure in the
NH2-terminal part of C-TERM may structurally support the B
subfragment as a backbone. Further, during the enzyme reaction of
topoisomerase II, a hinge motion of a polypeptide segment located
between the A
and B
subfragments is suggested by an SV8 proteolysis
experiment (38). The COOH-terminal part of C-TERM is expected to serve
as the hinge region because it corresponds to the disordered linker
region between the A
and B
subfragments in the crystal structure
(37). These features of C-TERM led us to the hypothesis that one of the
roles of this region is to provide a structure necessary for the
conformational change of gyrase. During the enzyme reaction, the B
subunits have to come apart for DNA entering. The contact to the other
B subunit must be lost in this step, and the hinge motion of the
COOH-terminal part of C-TERM is thought to separate each B subunit (37,
39), which is probably supported structurally by the
NH2-terminal part of C-TERM. Therefore, a mutation in
C-TERM is expected to cause a disordered arrangement of the whole B
subunit and/or disadvantage in conformational change of gyrase.
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1
The abbreviations used are: DTT, dithiothreitol;
BSA, bovine serum albumin.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.