From the Laboratoire de Génétique des
Procaryotes, Département de Biologie Moléculaire,
Université Libre de Bruxelles, rue des Chevaux 67, B-1640
Rhode-Saint-Genèse, Belgium, the
Laboratory of Molecular
Biology, NIDDK, National Institutes of Health, Bethesda, Maryland
20892-0540, and ** Dienst Ultrastruktuur, Vlaams Interuniversitair
Instituut voor Biotechnologie, Vrije Universiteit Brussel,
Paardenstraat 65, B-1640 St. Genesius-Rode, Belgium
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ABSTRACT |
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The F plasmid-carried bacterial toxin,
the CcdB protein, is known to act on DNA gyrase in two different ways.
CcdB poisons the gyrase-DNA complex, blocking the passage of
polymerases and leading to double-strand breakage of the DNA.
Alternatively, in cells that overexpress CcdB, the A subunit of DNA
gyrase (GyrA) has been found as an inactive complex with CcdB. We have
reconstituted the inactive GyrA-CcdB complex by denaturation and
renaturation of the purified GyrA dimer in the presence of CcdB. This
inactivating interaction involves the N-terminal domain of GyrA,
because similar inactive complexes were formed by denaturing and
renaturing N-terminal fragments of the GyrA protein in the presence of
CcdB. Single amino acid mutations, both in GyrA and in CcdB, that
prevent CcdB-induced DNA cleavage also prevent formation of the
inactive complexes, indicating that some essential interaction sites of
GyrA and of CcdB are common to both the poisoning and the inactivation
processes. Whereas the lethal effect of CcdB is most probably due to
poisoning of the gyrase-DNA complex, the inactivation pathway may
prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and
inactivation can be prevented and reversed in the presence of the F
plasmid-encoded antidote, the CcdA protein. The products of treating
the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA
complex of approximately 44 kDa, which may correspond to a
(CcdB)2(CcdA)2 heterotetramer.
The ability to modulate the topological state of DNA is essential
to bacteria. One of the enzymes responsible for this critical function
is DNA gyrase (a bacterial type II topoisomerase). In Escherichia
coli, DNA gyrase consists of two subunits,
GyrA1 and GyrB, of molecular
masses 97 and 90 kDa, respectively, encoded by the gyrA and
gyrB genes; the active enzyme is an
A2B2 complex. The A subunits mediate the
ability of the enzyme to introduce and rejoin double-strand breaks in
DNA. The B subunits mediate energy transduction and ATP hydrolysis (for
recent reviews, see Refs. 1-3).
Gyrase was first identified by its ability to convert relaxed circular
DNA into a negatively supercoiled form at the expense of ATP hydrolysis
(4). Given its role in the maintenance of proper levels of negative
supercoiling, and the overall role of supercoiling in transcription,
replication, and recombination (1, 2, 5), gyrase influences nearly all
DNA transactions in prokaryotic cells.
DNA gyrase is the target of several antibacterial agents, including the
coumarin and quinolone families of antibiotic drugs (6-9). The
coumarin antibiotics inhibit supercoiling activity by interfering with
ATP binding and hydrolysis. The quinolones block all activities of
gyrase that involve DNA strand passage. However, quinolone drugs tend
to exert their bactericidal action through complex formation with
gyrase and DNA rather than through simple elimination of gyrase
activity. The quinolones are thought to trap a reaction intermediate in
which gyrase has broken the phosphodiester backbone of DNA, while the
broken DNA ends are prevented from separation by binding to the enzyme.
This intermediate is often referred to as the "cleavable complex."
In this complex, the DNA is covalently joined to gyrase by phosphoryl
tyrosine bonds between the 5' ends of the cleaved DNA and
Tyr122 of the GyrA subunits (10). The quinolone drugs can
be considered to "poison" the DNA-gyrase complex by stabilizing the
DNA lesion. Double-strand breaks in the DNA are revealed upon
subsequent addition of a protein denaturant such SDS or alkali. Using
temperature-sensitive mutations, Kreuzer and Cozzarelli (11) showed
that growth of some bacteriophages is very sensitive to nalidixic acid
but not to the elimination of gyrase supercoiling activity; they
proposed that the gyrase-nalidixic acid complex on DNA could form a
barrier to the passage of polymerases. The observation of Willmott
et al. (12), that the gyrase-quinolone complex with DNA
leads to the blocking of transcription by E. coli and T7 RNA
polymerases in vitro, supports this hypothesis. In
vivo, it is thought that blocking of DNA polymerase may be the
critical event that leads to cell death. Killing appears to be caused
by events (subsequent to blockage of DNA replication) that may result
in release of the broken DNA ends from the cleavage complex (13).
Possible mechanisms that have been suggested for such a displacement of DNA ends from a drug-stabilized topoisomerase-DNA cleavable complex include dissociation of the gyrase tetramer (13), protein denaturation during an attempt to repair the DNA lesion (14), and helicase unwinding
of the DNA (15).
Naturally occurring protein inhibitors that cause
gyrase-dependent killing of E. coli with a mode
of action similar to that of the quinolone antibiotics have also been
found. Microcin B17 targets GyrB protein (16) and the CcdB protein
encoded by the E. coli F plasmid targets GyrA (17). The
ccdB gene is one partner of a two-gene system in the F
plasmid (together with ccdA) that results in the death of
cells that have lost the plasmid (18, 19). In F-containing cells both
genes are expressed, and CcdA protein (8.3 kDa) specifically blocks the
lethal action of CcdB (11.7 kDa). However, CcdA protein is
metabolically unstable due to its sensitivity to
Lon-dependent proteolysis (20). As a consequence, cells
that lose F ultimately retain only CcdB and are killed. Both CcdA and
CcdB are involved in the negative autoregulation of their own synthesis
at the transcriptional level (21, 22). Mutations in ccdB
that produce non-lethal proteins were found to cause amino acid
substitutions or deletions in the three C-terminal residues (23,
24).
Several lines of evidence have suggested that gyrase is the
intracellular target of CcdB protein. Independently isolated CcdB resistance mutations were found to map in the gyrA gene; one
of these mutants, A462, was sequenced
and found to produce an amino acid substitution of Arg462
to Cys (17). Separately, CcdB-tolerant mutations have been isolated in
several genes: gyrA, groES (mopB), and
groEL (mopA) (25); and tldD and
tldE (pmbA) (26). The role of the
groE, tldE, and tldD gene products is
still unclear but they have been proposed to act as factors implicated
in the interaction between the CcdB protein and DNA gyrase. Bernard and
Couturier (17) and Bernard et al. (27) have shown that CcdB
interacts with gyrase to cause double-strand breaks in DNA both
in vivo (which were revealed after SDS and proteinase K
treatment of lysates or of total DNA extracted from cells that express
plasmid-encoded CcdB in the absence of CcdA) and in vitro,
and have proposed that CcdB converts gyrase into a DNA poison in a
manner similar to the quinolone drugs by trapping gyrase in the
cleavable complex. The CcdA protein was able to prevent and to reverse
CcdB-induced DNA cleavage in vitro. Furthermore, gyrase
reconstituted with the purified CcdB-resistant mutant
GyrA462 subunit (see above) was refractory to CcdB-induced
DNA cleavage. Critchlow et al. (28) have demonstrated that,
like the quinolone drugs, CcdB forms a complex with gyrase and DNA that
can block transcription in an in vitro assay system.
Independently, Maki et al. (29, 30) examined DNA
supercoiling in cells overproducing the CcdB protein, and found that plasmid DNA in those cells was relaxed. GyrA protein purified from this
strain was found as a complex with CcdB, and was unable to catalyze DNA
supercoiling in the presence of GyrB protein. GyrB-dependent supercoiling activity of the GyrA in this
complex could be restored by addition of CcdA protein. These authors
suggested that the intracellular DNA relaxation was due to gyrase
inactivation by formation of the complex between GyrA and CcdB, and
that this process could be a cause of cell killing. However, they were
unable to reproduce this inactivation of gyrase by CcdB protein
in vitro. They concluded that gyrase inactivation and
topoisomerase poisoning by CcdB are two independent processes.
In this work, we have reconstituted the inactive GyrA-CcdB complex
in vitro, by denaturation-renaturation, and compared its properties with those of the complex made in vivo. We have
reactivated the complex by the addition of CcdA or of CcdA41, a 41- residue C-terminal fragment of CcdA that has lost its autoregulatory
properties but retains the ability to reverse the lethal effects of
CcdB (31), and characterized the products of the reactivation
procedure. We have also investigated the CcdB-resistant
GyrA462 protein (17), the truncated GyrA 64- and 59-kDa
N-terminal domain proteins (28, 32, 33), and the non-lethal
CcdBo15 mutant protein (23), all of which are unable to
participate in CcdB-mediated cleavage of DNA, for their ability to
replace wild-type GyrA or CcdB in the formation of the inactive complex.
Bacterial Strains and Plasmids--
MAX Efficiency
DH5 Gyrase Purification and Assays--
DNA gyrase was reconstituted
from individual subunits, which were purified as was described by
Mizuuchi et al. (38). The specific activities of gyrase A
and B subunits, determined in DNA supercoiling assays, were 3.5 × 106 and 5 × 105 units/mg of protein,
respectively. The GyrA462 protein was isolated from a
JM109/pPH310 strain according to the same protocol. Its specific
activity was 4 × 106 units/mg of protein. The
N-terminal 64- and 59-kDa fragments of GyrA were expressed from pRJR242
and pRJR10.18 by isopropyl-1-thio- Preparation of the CcdA, CcdB, and CcdBo15
Proteins--
CcdA protein was produced in CSH50
lon::Tn10 carrying the pULB2709 CcdA-overproducing
plasmid (27), and purified as described (39). CcdA41 was synthesized by
A. Tartar, Institut Pasteur de Lille (France).
CcdB and CcdBo15 (23) proteins were expressed in a host
strain carrying the CcdB resistance mutation
gyrA462 and purified as described (40) with the
following modifications: After ammonium sulfate precipitation and
resuspension in buffer A (50 mM Tris, pH 8.0), the
precipitated proteins were dialyzed against buffer A and applied to a
MonoQ anion exchange column equilibrated with the same buffer. The
fractions containing CcdB or CcdBo15 were pooled, dialyzed
overnight against 25 mM MOPS, pH 7.0, and loaded onto a
MonoS cation exchange column equilibrated in the same buffer. The
elution yielded about 30 mg of pure CcdB or CcdBo15/liter
of cell culture.
Gel Filtration and Polyacrylamide Gel
Electrophoresis--
Analytical gel filtrations were performed using a
Superdex 200 PC 3.2/30 precision column on a Smart System (Amersham
Pharmacia Biotech) in a buffer containing 50 mM Tris-HCl,
pH 7.5, 1 mM EDTA, 700 mM NaCl, and 10% (v/v)
glycerol, at a flow rate of 50 µl/min, or using a Superdex 75 HR
(10/30) FPLC column in 20 mM Tris-HCl, pH 8, 150 mM NaCl at 1 ml/min. Molecular weight markers used to calibrate these columns were: ferritin (440,000), catalase (232,000), aldolase (158,000), bovine serum albumin (67,000), ovalbumin (43,000), chymotrypsinogen A (25,000), and ribonuclease A (13,700); or a gel
filtration standard mixture (Bio-Rad) containing gamma globulin (158,000), ovalbumin (44,000), myoglobin (17,000), and vitamin B12
(1,350). Polyacrylamide gel electrophoresis in the presence of SDS was
done using 4-20% Tris-glycine or 10-20% Tricine gradient gels and
Tris-glycine-SDS or Tricine-SDS running buffers obtained from Novex.
Non-denaturing gel electrophoresis was done using Novex 6%
polyacrylamide gels in TBE or in Tris-glycine native running buffers.
In Vitro Binding of CcdB to the GyrA Protein and Purification of
the Complex--
The inactive complex between CcdB and GyrA was formed
by denaturation/renaturation, by mixing GyrA (60 µg/ml) and an 8-fold molar excess of CcdB (60 µg/ml) in buffer consisting of 5 M urea in 35 mM Tris-HCl, pH 7.5, 30 mM NH4Cl, 0.1 mM EDTA, 5 mM dithiothreitol, and 10% glycerol. After incubation on
ice for 30 min, the mixture was dialyzed overnight at 4 °C against
the same buffer without urea. Purification of the inactivated GyrA-CcdB
complex from free GyrA and free CcdB was carried out using a Hi-Trap
Heparin column (Amersham Pharmacia Biotech). The column was washed with
five column volumes of equilibration buffer (10 mM sodium
phosphate, pH 7.0, 10% glycerol, 0.1 mM EDTA, 5 mM dithiothreitol) and eluted with a 20-column volume
linear gradient of 0.0-1.0 M NaCl in the equilibration
buffer. The protein composition of each peak was confirmed by
SDS-polyacrylamide gel electrophoresis, and the peak containing the
inactive complex was further characterized by
GyrB-dependent supercoiling assays performed in the
presence and absence of CcdA.
Complexes between the N-terminal GyrA 64- or 59-kDa proteins and CcdB
were also prepared by denaturation in the presence of urea, followed by
renaturation upon dialysis, as described above.
Under non-denaturing conditions, a CcdB complex with the GyrA 59-kDa
protein was prepared by slowly adding 195 µl of a 3 mg/ml solution of
CcdB to 1.33 ml of a 1 mg/ml GyrA59 solution in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, at
14.1 °C, over a period of 45 min (15 injections of 13 µl each,
with a 3-min interval between additions). This complex was separated
from free CcdB on a Superdex 75 HR (10/30) column in the same buffer.
Purification of the GyrA-CcdB Complex Formed in
Vivo--
Production and purification of the in vivo
GyrA-CcdB complex was performed as described (30), with the following
modifications. In order to obtain a large amount of inactivated GyrA,
while allowing a better bacterial growth rate, we have used the pPH31
plasmid, which overproduces GyrA protein and supplies the
LacIq repressor to regulate the CcdB and GyrA protein
production. CcdB protein was co-produced from pMLOccdB.
DH5 Reactivation of the GyrA-CcdB Complex with CcdA and
CcdA41--
Reconstituted GyrA-CcdB complex, which was obtained by
denaturation-renaturation and purified by Hi-Trap Heparin
chromatography (20 µg), and CcdA protein (10 µg) or CcdA41 (10 µg) were incubated for 5 min at room temperature in 100 µl of 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 700 mM NaCl, and 10% (v/v) glycerol. The mixture was then
resolved on a Superdex 200 gel filtration column in the same buffer, at
a flow rate of 50 µl/min.
In Vitro Formation of the CcdA-CcdB Complex on a
GyrA59-Sepharose 4B Column--
The truncated gyrase A
59-kDa N-terminal protein was coupled to CNBr-activated Sepharose 4B
(Amersham Pharmacia Biotech), at 6 mg of GyrA59/ml of
CNBr-Sepharose, according to the manufacturer's instructions. A 1 ml
FPLC column was prepared and equilibrated at room temperature with 20 mM Tris, pH 8, 150 mM NaCl prior to loading 10 ml of 0.55 mg/ml CcdB in the same buffer. The column was extensively
washed with equilibration buffer to remove all free CcdB, and then
eluted with 10 ml of 0.3 mg/ml CcdA in the same buffer. The eluted
CcdA-CcdB complex was further fractionated using a Superdex 75 HR
(10/30) column (Amersham Pharmacia Biotech) in the same buffer, and
fractions were analyzed by SDS-PAGE.
In Vivo Production and in Vitro Reconstitution of the GyrA-CcdB
Inactive Complex--
We have reconstituted an inactive GyrA-CcdB
complex in vitro by denaturation and renaturation of
purified GyrA in the presence of excess CcdB. In order to directly
compare the purification behavior and the physical and catalytic
properties of this reconstituted complex with those of the previously
reported complex formed in vivo, we also purified the
inactive GyrA-CcdB complex from a strain overproducing GyrA and CcdB
proteins according to a protocol similar to that described (30) (see
"Experimental Procedures"). At the last step of this purification,
a Hi-Trap Heparin column, two separate peaks were obtained late in the
gradient; the first at 0.65 M NaCl corresponded to free
GyrA and the second at 0.7 M NaCl to the inactive GyrA-CcdB
complex. The identity of each peak was confirmed by SDS-polyacrylamide
gel electrophoresis. The in vitro complex between GyrA and
CcdB was obtained as described under "Experimental Procedures."
When a portion of the renatured material was subjected to Superdex 200 gel filtration, two peaks were found (Fig.
1A). SDS-PAGE analysis
revealed that the peak that eluted first contained both GyrA and CcdB;
the second peak was free CcdB (Fig. 1B). The remainder of
the renatured sample was then subjected to Hi-Trap Heparin column
chromatography. Late in the gradient, only one peak was obtained, at
0.7 M NaCl, which corresponds to the GyrA-CcdB complex;
free GyrA was not detected.
The inactive GyrA-CcdB complexes produced in vitro and
in vivo co-migrate in native gels at a position different
from those of the free GyrA and CcdB proteins (data not shown) and
exhibit a 90-95% inhibition of GyrB-dependent DNA
supercoiling activity (Fig. 2; Refs. 29
and 30). The strong resemblance between the in vivo and
in vitro complexes indicates that the complex reconstituted
in vitro is equivalent to that formed in vivo in CcdB-overproducing strains. No inactive complex and no reduction of
GyrA-dependent supercoiling activity could be detected in
in vitro control samples where the urea treatment was
omitted.
Inactive complexes between the GyrA N-terminal 64- or 59-kDa protein
fragments and CcdB were also formed during a denaturation and
renaturation procedure. Although these truncated GyrA proteins do not
perform efficient ATP-dependent DNA supercoiling in the presence of GyrB protein, they do participate in GyrB- and
ATP-dependent relaxation of supercoiled DNA (33); this
relaxation activity was inhibited when the CcdB-inactivated truncated
proteins were assayed (Fig. 3). The
inactive complexes of GyrA64 and GyrA59 with
CcdB migrated on native gels at positions intermediate between those of
the free proteins (data not shown).
Interestingly, complexes of CcdB with the truncated GyrA proteins
appear to form spontaneously, since bands on native gels that
correspond to these complexes were found in the control samples, which
were not denatured with urea. An in vitro experiment with the GyrA 59-kDa protein and CcdB under nonchaotropic conditions confirmed this observation, and the highest yield of complex was obtained after a slow titration of CcdB into a GyrA59
solution (Fig. 4).
In Vitro Reactivation of the Reconstituted GyrA-CcdB Complex by
CcdA and CcdA41--
Fractions of the GyrA-CcdB complex reconstituted
in vitro (0.7 M NaCl Hi-Trap heparin column
peak) were used to perform GyrB-dependent supercoiling
assays in absence or presence of CcdA (Fig. 2). In the absence of CcdA,
approximately 10% of GyrA activity persists in the complex compared
with the free GyrA used as a control. Similar supercoiling activity has
been reported for the GyrA-CcdB complex produced in vivo
(29, 30). This residual activity may be due to dissociation of the
complex during the assay. In the presence of CcdA, the
GyrB-dependent supercoiling activity was entirely
recovered. CcdA can also restore ATP- and GyrB-dependent DNA relaxing activity to the inactive CcdB complexes in which the
truncated GyrA 64- and 59 kDa N-terminal proteins are substituted for
the full-length GyrA protein (Fig. 3).
CcdA41, which possesses only the C-terminal 41 amino acids of CcdA, can
also neutralize the toxicity of CcdB in vivo (31). Using
purified CcdA protein or a synthetic CcdA41, we investigated the
ability of both proteins to remove CcdB from the GyrA-CcdB complex, and
to form stable complexes with this extracted CcdB protein. After a
brief incubation of CcdA or CcdA41 with the GyrA-CcdB complex, the
mixtures were resolved on Superdex 200 gel filtration columns. The gel
filtration profiles show that when the GyrA-CcdB complex was run alone,
only one peak, corresponding to this GyrA-CcdB complex, was obtained.
However, when either CcdA or CcdA41 was added in excess over CcdB to
the complex, three peaks were obtained (Fig.
5A). Each peak was analyzed on
a polyacrylamide denaturing gel (Fig. 5B). The first peak corresponds
to free GyrA and contains no CcdB; the second peak contains the newly
formed CcdB-CcdA or CcdB-CcdA41 complex; the third peak corresponds to
the free CcdA or CcdA41. By comparing the positions of the elution
peaks of the complexes with those of protein size standards run on the same Superdex 200 column, the CcdB-CcdA complex shows an estimated molecular weight of 44,000, close to that expected for a tetrameric complex consisting of a dimer of CcdB and a dimer of CcdA. The CcdB-CcdA41 complex has an estimated molecular weight of 29,000 which
is near that of a tetrameric complex comprising a dimer of CcdB with a
dimer of CcdA41. The peaks of free CcdA and free CcdA41 correspond
closely to dimers of molecular weight 20,000 and 9,000, respectively.
A similar result was obtained when the CcdB-GyrA 59-kDa protein complex
was formed under non-denaturing conditions on a
GyrA59-Sepharose 4B column, followed by elution of the CcdB
from the complex with an excess of CcdA. Upon fractionation of the
eluted material by Superdex 75 gel filtration, we found one peak
corresponding to the tetrameric complex
(CcdA)2(CcdB)2 with a molecular weight of
41,000, and a second peak corresponding to (CcdA)2 with a
molecular weight of 20,000 (Fig. 6).
These observations demonstrate that CcdB is quickly and completely
extracted from a GyrA-CcdB complex by CcdA and by CcdA41 and that new
CcdB-CcdA or CcdB-CcdA41 complexes are formed. Thus, the affinity of
CcdB for CcdA or CcdA41 is apparently greater than its affinity for
GyrA.
Mutations in the GyrA and CcdB Proteins Which Prevent Poisoning of
the Cleavable Complex Also Prevent GyrA Inactivation--
CcdB has
been shown to trap DNA gyrase in a cleavable complex such that, on
addition of SDS or alkali, double-strand cleavage of DNA is observed
(17, 27). An important question arises: are the same interaction sites
of GyrA and CcdB implicated in this poisoning of the DNA-gyrase complex
and in the inhibition of GyrB-dependent supercoiling
activity of the GyrA subunit? The GyrA462 mutant protein,
which confers resistance to CcdB in vivo (17), and
CcdBo15, a non-killer CcdB protein (23), were used to
perform CcdB-mediated cleavage reactions. (The mutation in
ccdB which produces CcdBo15 was previously
reported to be a TA to AT transversion resulting in the replacement of
Ile101 by Lys (23). Upon resequencing of the plasmid encoding this
mutant protein, we found instead a GC to CG transversion that replaced
Trp99 with Ser, the same mutation as was found in
CcdBo14 (23).) GyrA462, in the presence of
GyrB, is unable to promote any CcdB-mediated DNA cleavage (Ref. 27;
Fig. 7). Similarly, when
CcdBo15 was assayed for in vitro DNA cleavage in
presence of GyrA and GyrB, no DNA cleavage was observed (Fig. 7). These
mutant proteins were then tested for their ability to form inactive
complexes (GyrA462-CcdB, GyrA-CcdBo15, or
GyrA462-CcdBo15) in vitro. After
denaturation and renaturation, the protein solutions were resolved on
Superdex 200 gel filtration columns (see Fig. 1A). Fractions
from each peak were evaluated by denaturing gel electrophoresis. The
complex was formed only when the wild type GyrA and CcdB proteins were
used together; with all the other combinations, no complex was formed
(Fig. 1B) and no inhibition of GyrB-dependent
supercoiling was found (data not shown). Attempts to produce these
complexes in cells overexpressing CcdBo15 with wild-type
GyrA or wild-type CcdB with GyrA462 were also unsuccessful,
as no complexes could be detected. From these results, we conclude that
the protein interactions between CcdB and gyrase in the cleavable DNA
complex and between CcdB and GyrA in the inactive GyrA-CcdB complex
share essential sites.
It is well established that the intracellular target for the F
plasmid toxin, the CcdB protein, is DNA gyrase (17, 25-30). However,
neither its mode of interaction with gyrase nor the manner in which
this interaction leads to cell death is yet fully understood.
Two different hypotheses are currently considered. The first suggests
that CcdB is a poison of the DNA-gyrase cleavable complex in a manner
similar to the quinolone drugs. Bernard and co-workers (17, 27) have
found that DNA cleavage by gyrase-CcdB complexes can be revealed both
in vitro in the presence of ATP and SDS, and in
vivo by SDS treatment of whole cells. Furthermore, when complexed
with GyrB, the GyrA462 protein (a mutation that confers resistance to CcdB in vivo) cannot support CcdB-induced
cleavage of DNA in vitro. In the
gyrA462 strain, no CcdB-dependent
plasmid linearization was observed after SDS treatment of the cells. It was also observed that the CcdB-sensitive phenotype dominates over the
resistant phenotype in a partial diploid strain. This phenomenon, where
sensitivity is dominant over resistance, is also observed with
quinolone drugs (7, 41) and indicates that CcdB probably converts
wild-type gyrase into a poison.
The second hypothesis considers CcdB protein as an inhibitor of
gyrase-mediated DNA supercoiling. Increased intracellular expression of
GyrA protein has been found to oppose growth inhibition caused by CcdB
(25). A second CcdB-resistant mutant was isolated and shown to have a
different point mutation in the gyrA gene, which results in
an amino acid change of Gly to Glu at residue 214 of the GyrA protein
(25). This mutant GyrA was found to be trans-dominant over wild-type
GyrA protein. Analysis of DNA supercoiling in bacteria overproducing
the CcdB protein (29, 30) showed that the DNA was extensively relaxed
(around 99% of DNA was relaxed upon
isopropyl-1-thio- In considering these hypotheses, we have investigated whether the
inactivation of GyrA in the GyrA-CcdB complex occurs through a
different interaction than that which traps a covalently linked gyrase-DNA complex in the poisoning process. We first purified the
inactive complex made in vivo, from a strain overproducing both GyrA and CcdB. In vitro, CcdB binding to GyrA does not
occur spontaneously, and it has been suggested that, within the cell, additional factors such as the groE, tldD, and
tldE gene products may be involved (25, 26). We have
reconstituted the inactive GyrA-CcdB complex from the purified proteins
in vitro by denaturation in urea, followed by renaturation
upon dialysis. The complex reconstituted in vitro was
compared with the one made in vivo; all of their properties
are similar.
Using purified CcdB-resistant GyrA462 protein, and the
non-lethal CcdBo15 protein, we investigated whether these
two mutants, which are defective for CcdB-induced DNA cleavage, are
also defective for GyrA inhibition. When GyrA462 or
CcdBo15 were separately substituted for their wild-type
counterparts in CcdB-mediated DNA cleavage reactions, no DNA
linearization was observed (Fig. 7), confirming that these two
mutations each prevent the CcdB interaction with gyrase in the
poisoning process. These two mutated proteins were then tested for
complex formation in vitro by denaturation-renaturation. No
GyrA462-CcdB or GyrA-CcdBo15 complexes were
obtained (Fig. 1B). We conclude that at least some of the
same interaction sites between GyrA and CcdB are essential both for DNA
cleavage and for GyrA inactivation.
However, these sites participate in the formation of two different
complexes: a ternary complex (poison complex) consisting of the gyrase
holoenzyme, DNA, and CcdB protein, and a binary complex (inhibition
complex) between CcdB and the GyrA protein. The ternary complex leads
to nucleotide-dependent DNA breakage when gyrase is bound
on the DNA. In this case the gyrase conformational changes involved in
ATP binding and hydrolysis, and in opening the DNA gate for the passage
strand translocation of the supercoiling cycle, may be necessary to
allow the CcdB access to its site of interaction on the GyrA. CcdB
binding then may prevent religation of the DNA, leading to its
breakage. Critchlow et al. (28) have suggested that CcdB may
bind to a post-translocation intermediate in the DNA supercoiling
reaction. Because of the transient nature of such an intermediate,
formation of the ternary complex would probably be quite inefficient;
the majority of the gyrase-DNA cleavable complexes would not be
trapped, and the observed gyrase supercoiling activity would not be
appreciably inhibited. However, the presence of only a very few
CcdB-stabilized chromosomal DNA lesions should be sufficient to poison
a bacterial cell. As is seen with the quinolone drugs in
vivo, CcdB induces the SOS response, indicative of DNA damage (17,
42), and CcdB induction of SOS appears to require the helicase (but not
the nuclease) activity of RecBCD (43). Also as with quinolones, the
CcdB-poisoned complex, in addition to causing DNA cleavage, can form a
barrier to the passage of polymerases (28). However, in
vitro, CcdB-induced DNA cleavage is
nucleotide-dependent, whereas quinolone-induced DNA
cleavage is not. Furthermore, CcdB-induced DNA cleavage is not
reversible by methods known to reverse quinolone-induced cleavage, such
as treatment by heat, salt, or EDTA. Thus, it is thought that the
quinolone drugs and CcdB may stabilize different conformations of the
cleavage complex intermediate in the gyrase topoisomerase reaction
cycle (28, 44). A ternary complex between DNA, gyrase, and CcdB has
recently been assembled in vitro and purified (44).
Formation of the inhibition complex causes inactivation of free GyrA
protein, which is then unable to participate in supercoiling or
cleavage of DNA (30). The requirement for denaturation to allow
GyrA-CcdB binding in vitro indicates that the CcdB
interaction site on GyrA is protected in the native GyrA dimer.
Intracellular formation of this inactive GyrA-CcdB complex may be due
to binding of CcdB to newly translated GyrA monomers that have not yet
folded and formed dimers. In vivo, DNA in CcdB-overproducing
cells is relaxed (29, 30) and heat-shock proteins are induced (24). Intracellular DNA relaxation and heat-shock protein induction are also
characteristic of the coumarin antibiotics (45-47).
The manner in which the interaction of CcdB with gyrase causes cell
death has been unclear. As discussed above, gyrase-DNA complex
poisoning by CcdB and GyrA inactivation by CcdB appear to be closely
related. Both are prevented or reversed by the antidote CcdA protein;
under different conditions of CcdB expression, both can be demonstrated
to occur in vivo; and each displays intracellular effects
similar to those of one of the two major families of antibiotics that
target gyrase. However, using a system that simulates the killing of
bacteria that have lost F, Bernard and Couturier (17) examined the DNA
of CcdB-expressing cells in which synthesis of CcdA was conditionally
suppressed. These cells showed only a slight decrease in plasmid DNA
supercoiling. When the total DNA of the SDS-treated cells was analyzed,
double-strand breaks were found in the bacterial chromosome and in a
resident plasmid. These observations indicate that stabilization of a
DNA lesion by CcdB may be essential for cell killing, whereas DNA
relaxation is not. They strongly suggest that the lethal effect of CcdB
protein on bacterial cells occurs through poisoning of the gyrase-DNA
complex rather than by inhibition of supercoiling. This model is also
supported by the ability of the gyrase-DNA-CcdB complex to block the
passage of polymerase (28).
What, then, may be the intracellular role of the GyrA-CcdB complex?
Significant relaxation of intracellular DNA would require that most of
the GyrA in the cell would be found in the CcdB complex; this would be
unlikely at normal expression levels of CcdB. Moreover, expression of
the gyrase proteins is regulated by the topological state of the DNA
and so a decrease in supercoiling would result in increased gyrase
synthesis (46) to maintain the normal supercoiling density. Perhaps the
most relevant function of this complex may be, not inactivation of GyrA
to inhibit DNA supercoiling, but inactivation of CcdB to prevent
polymerase blocking and DNA cleavage. We propose that the GyrA-CcdB
complex may function as an antidote-poison combination, which prevents
stabilization of DNA lesions by neutralizing the CcdB toxin. Formation
of this complex between CcdB and newly synthesized GyrA could be the
means by which increased intracellular expression of GyrA overcomes
CcdB-induced growth inhibition, as has been shown elsewhere (25). This
may protect F plasmid-containing bacteria against transient small
intracellular excesses of CcdB over CcdA, which may occur in response
to environmental perturbations liable to activate proteolysis of CcdA.
Thus, the GyrA-CcdB complex may contribute to cell survival instead of
causing death.
In previous work, it was found that, in vitro, the GyrA
N-terminal 64-kDa domain did not allow CcdB-mediated DNA cleavage in
the presence of GyrB (28). One possible explanation was that the
removal of the GyrA C-terminal 33-kDa domain removed an essential component of the CcdB binding site, even though the CcdB resistance mutations of GyrA are all found in the N-terminal 64-kDa domain. Gyrase
reconstituted using the GyrA 64- or 59-kDa N-terminal fragments is no
longer able to negatively supercoil the DNA; however, it has an
ATP-dependent DNA relaxing activity characteristic of all other type II topoisomerases (33). Our denaturing-renaturing procedure,
using CcdB and the GyrA 64- or 59-kDa N-terminal proteins, produces
complexes that, in the presence of GyrB, have greatly decreased
ATP-dependent DNA relaxation activities. DNA relaxation is
substantially restored in the presence of CcdA (Fig. 3). When control
complex-formation reactions were done in parallel, under non-denaturing
conditions (no urea), full-length GyrA did not form a complex with
CcdB; however, both GyrA64 and GyrA59 did form
complexes with CcdB, which co-migrate on native gels with the
corresponding complexes obtained by denaturation and renaturation (data
not shown).
Together, these findings indicate that CcdB binding to GyrA is not
dependent on sites located in the C-terminal 33-kDa domain of GyrA or
on residues that are protected in the GyrA primary dimer interface. It
should be noted that, in the crystal structure of the GyrA 59-kDa
N-terminal domain (48) the Arg462 residue is located in the
central cavity of the GyrA dimer, near but not in the primary dimer
interface. Structural data for the C-terminal 33-kDa domains of the
GyrA subunits, in relation to the rest of the GyrA dimer, is not yet
available. However, based on the above observations, it is likely that
the GyrA C-terminal 33-kDa domains, which are thought to play a part in
the wrapping of DNA around the gyrase tetramer (49), are contributing
factors in blocking free access of CcdB to binding sites on full-length native GyrA.
It would be reasonable to assume that the inability of the
GyrA64- or GyrA59-substituted gyrase to
participate in the CcdB-mediated DNA cleavage reaction might be due to
their inactivation by formation of complexes with CcdB. However, in the
absence of denaturation, complex formation is quite slow (incorporation
of GyrA59 into a complex with CcdB is approximately 50%
after 20 h at 4 °C, or 75% after 4 h at 37 °C; data
not shown), and should not completely account for the loss of
CcdB-mediated cleavage activity under the conditions of the assay. It
is more likely that gyrase reconstituted using the truncated GyrA
proteins is unable to bind and present the DNA in the required
conformation of the intermediate target for CcdB to form the ternary
(poisoned) cleavage complex, as has been suggested (28).
We have demonstrated that, when CcdA or CcdA41 are used to remove CcdB
from the inactive GyrA-CcdB complex, the products correspond to the
equimolar heterotetramers (CcdA)2(CcdB)2 and
(CcdA41)2(CcdB)2. However, when purified CcdA
and excess CcdB were mixed in vitro, a higher molecular
weight CcdA-CcdB complex was reported (39). This complex had an
estimated molecular weight of about 60,000, close to that expected for
a complex comprising a tetramer of CcdB with a dimer of CcdA. Upon
titration of CcdA protein with increasing levels of CcdB and analysis
by native gel electrophoresis, a diffuse band representing a species
with an equimolar ratio of CcdA to CcdB was found when the
concentration of CcdA exceeded or equaled that of CcdB; increasing
levels of the Mr 60,000 species were obtained
with an increasing molar excess of CcdB, and free CcdB did not appear
until the CcdB:CcdA ratio exceeded 2:1 (39). Tam and Kline (21) have
found a CcdA-CcdB complex in lysates of a strain that overproduces the
two proteins. The molecular weight of this complex was estimated to be
about 69,000. A protein complex of the same size was also obtained by
stripping the Ccd proteins from their complex with a plasmid containing
the cloned ccd operator (21). This suggests that the 60,000 or 69,000 CcdA-CcdB complex may mediate autoregulatory functions,
whereas the 44,000 (CcdA)2(CcdB)2 tetramer
obtained in our reactivation procedure may represent the antidote-toxin complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F'IQTM competent E. coli cells were from
Life Technologies, Inc. E. coli JM109 (34) was from New
England Biolabs. The plasmid pPH3 (35) containing the
lacIq gene and the gyrA gene under
control of the tac promoter was a gift from A. Maxwell. This
plasmid has two PstI sites, one inside the gyrA
gene and the other one in the vector. To remove the one in the vector,
the plasmid was partially digested with PstI restriction enzyme, blunt-ended with the Klenow fragment of DNA polymerase I, and
religated with T4 DNA ligase. The plasmid that has lost the vector
PstI site was called pPH31. The
KpnI-PstI gyrA fragment from pPH31 was
replaced by the same fragment from pULB2229 (17) containing the
gyrA462 mutation and was called pPH310. Plasmids pRJR242 and pRJR10.18 (32), which express GyrA N-terminal 64- and
59-kDa fragments, respectively, were also gifts from A. Maxwell. The
pMLOccdB plasmid was constructed by cloning the
BamHI fragment from pULB2250 plasmid (27) containing the
ccdB gene under control of the tac promoter into
the BamHI site of
pMLO59,2 which was derived
from pGB2ts (36), a mini-pSC101 vector thermosensitive for replication.
Plasmid pMP1625 (27), which contains a preferred gyrase DNA
cleavage site on a BamHI-ClaI fragment of
bacteriophage Mu (37) cloned at the BamHI site of pBR322,
was used to perform cleavage assays. Relaxed or negatively supercoiled
pBR322 was used in DNA supercoiling or relaxation assays, respectively.
-D-galactopyranoside induction in DH5
F'IQTM and purified to near homogeneity
by ammonium sulfate precipitation followed by chromatography on Hi-Trap
Heparin columns (Amersham Pharmacia Biotech). Supercoiling assays were
performed as described by Mizuuchi et al. (38). CcdB and
oxolinic acid-induced cleavage of DNA was performed using
PstI-digested linear pMP1625 plasmid as substrate, under
supercoiling conditions and under the CcdB cleavage conditions of
Bernard and Couturier (17), and Bernard et al. (27). CcdB or
oxolinic acid (Sigma) were added at 1 and 0.05 mg/ml, respectively, and
ATP was either omitted or added at 1.4 mM. Cleavage assays
were terminated by addition of 0.2% SDS and 0.08 mg/ml proteinase K
(Beckman) and further incubated for 30 min at 37 °C. DNA relaxation
assays were performed under the reaction buffer conditions described
for CcdB-mediated cleavage (27), but the final incubation with SDS and
proteinase K was omitted.
F'IQTM, which also produces the LacIq
repressor, was used as the host strain.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Complex formation between wild-type and
mutant GyrA and CcdB. GyrA or GyrA462 was denatured
and renatured in the presence of CcdB or CcdB°15 as described under
"Experimental Procedures." Each mixture was resolved by gel
filtration on a Superdex 200 column eluted with 50 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 700 mM NaCl, and
10% (v/v) glycerol. A, the elution profile shown is typical
of that obtained with each of the above combinations. B,
complex formation was analyzed by subjecting both peaks from each
profile to denaturing gel electrophoresis on a 4-20% polyacrylamide
gradient gel run in Tris-glycine-SDS buffer and silver-stained. The
control sample was a mixture of free GyrA and free CcdB proteins.
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Fig. 2.
Supercoiling activity of the reconstituted
GyrA-CcdB complex in the absence or presence of CcdA. The DNA
supercoiling assay was performed as described (38). The 70-µl
reactions contained the following proteins: GyrB (20 ng,
lanes 2-9), GyrA (2 ng, lane
2), GyrA-CcdB complex (10 ng, lanes 3 and 6; 50 ng, lanes 4 and
7; 100 ng, lane 8; 200 ng,
lanes 5 and 9), and CcdA (200 ng,
lanes 6-9). Reactions were incubated at 25 °C
for 1 h, then terminated by addition of EDTA and SDS to final
concentrations of 10 mM and 0.5%, respectively. Product
DNA was analyzed on a 1% agarose gel run in TBE buffer.
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Fig. 3.
Inactivation of GyrA N-terminal 64- and
59-kDa domains by CcdB; reactivation by CcdA. DNA relaxation assay
buffer conditions were the same as the conditions of the cleavage
reactions described in the legend to Fig. 7. Truncated GyrA proteins
were first denatured and renatured in the absence ( ) or presence (+)
of CcdB as described under "Experimental Procedures."
GyrA64(
CcdB) (110 ng, lane 2; 220 ng, lane 3), GyrA64(+CcdB) (110 ng,
lanes 4 and 6; 220 ng,
lanes 5 and 7), GyrA59
(
CcdB) (27 ng, lane 9; 54 ng, lane
10), GyrA59(+CcdB) (27 ng, lanes
11 and 13; 54 ng, lanes 12 and 14), and CcdA (2.8 µg, lanes 6,
7, 13, 14, and 15) were
incubated at 30 °C for 30 min in 70 µl of reaction buffer lacking
ATP. GyrB protein (50 ng, lanes 9, 11,
and 13; 100 ng, lanes 2, 4,
6, 10, 12, and 14; 200 ng,
lanes 3, 5, and 7),
negatively supercoiled pBR322 DNA (0.7 µg, lanes
1-7 and 9-15), relaxed pBR322 DNA (0.7 µg,
lane 8), and ATP (1.4 mM,
lanes 1-15) were then added, and incubation was
continued at 30 °C for 2 h. After removal of protein, the
samples were analyzed on an 0.8% agarose gel run in TBE buffer.
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Fig. 4.
Complex formation between the GyrA 59-kDa
N-terminal domain protein and CcdB under non-chaotropic
conditions. A, after a slow titration of a molar excess
of CcdB into a GyrA59 solution in 20 mM
Tris-HCl, pH 8.0, 150 mM NaCl, the sample was resolved on a
Superdex 75 HR (10/30) gel filtration column in the same buffer.
B, protein peak fractions were analyzed by SDS-PAGE.
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Fig. 5.
Protein species present after reactivation of
the reconstituted GyrA-CcdB complex. After preincubation of the
reconstituted GyrA-CcdB complex (20 µg) with CcdA (10 µg) or
CcdA41 (10 µg) for 5 min at room temperature, the mixture
was passed through a Superdex 200 gel filtration column equilibrated
with 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 700 mM NaCl, and 10% (v/v) glycerol, at a flow rate of 50 µl/min. A, elution volume positions of the proteins are
indicated by the arrows on the gel filtration profiles.
Profile 1 corresponds to the GyrA-CcdB complex
before reactivation. Profiles 2 and 3 correspond
to the GyrA-CcdB complex reactivated with CcdA or CcdA41,
respectively. B, each peak in profiles
1 and 2 was analyzed on a 4-20% polyacrylamide
gradient gel run in Tris-glycine-SDS buffer and silver-stained.
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Fig. 6.
Complex formation between CcdA and CcdB on a
GyrA59 column. A 1.0-ml GyrA59-Sepharose
4B FPLC column was charged with CcdB and subsequently eluted with CcdA.
A, the eluted CcdA-CcdB complex was further fractionated on
a Superdex 75 HR (10/30) column in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl. B, peak fractions were analyzed by
SDS-PAGE.
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Fig. 7.
CcdB-mediated DNA cleavage: comparison of
wild-type and mutant gyrase and CcdB, and reversal by CcdA or
CcdA41. Linear DNA (PstI-restricted pMP1625, 0.7 µg)
was incubated with gyrase (140 ng, lanes 7-12)
or with gyrase reconstituted using GyrA462 (140 ng,
lanes 13-15) in 70 µl of buffer containing 35 mM Tris-HCl (pH 7.5), 7 mM MgCl2,
30 mM NH4Cl, 1.4 mM ATP, 0.36 mg/ml
bovine serum albumin, 35 µg/ml tRNA, 5 mM dithiothreitol,
1.5% glycerol. After 1 h at 25 °C, CcdB (1 µg,
lanes 2, 9-11, and 14) or
CcdBo15 (1 µg, lanes 3 and
12) or oxolinic acid (50 µg/ml, lanes
6, 8, and 15) was added and the
samples were incubated for 30 min at 30 °C. CcdA (2 µg,
lanes 4 and 10) or CcdA41 (2 µg,
lanes 5 and 11) was then added, and
incubation was continued at 30 °C for an additional 30 min. The
reactions were stopped by the addition of SDS and proteinase K and
further incubation at 37 °C for 30 min. After removal of protein,
the samples were analyzed on 0.9% agarose gels run in TBE
buffer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside induction of the
ccdB gene). The GyrA protein purified from this CcdB-overproducing strain was found as a complex with CcdB. In the
presence of GyrB and ATP, the GyrA-CcdB complex had little or no
supercoiling activity; however, GyrB-dependent supercoiling activity was recovered upon addition of CcdA. Surprisingly, this GyrA-CcdB complex was also inactive in both CcdB-mediated and quinolone-mediated DNA cleavage assays. The authors suggested that the
CcdB-mediated inhibition of supercoiling by gyrase might be involved in
cell killing. DNA supercoiling is a major determinant of cellular
functions; DNA replication, transcription, and recombination all depend
on the appropriate level of supercoiling being maintained in the cell.
Thus, inhibition of supercoiling could lead to cessation of cell
growth; however, this inhibition would not necessarily lead to cell death.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Maxwell for strains and plasmids and for experimental protocols on GyrA-Sepharose affinity column chromatography, and Dr. A. Tartar for CcdA41 protein.
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FOOTNOTES |
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* This work was supported in part by the Fondation Moeurs-François, the Fonds National de la Recherche Scientifique, the Fonds National de la Recherche Scientifique Médicale, the FNRS-Télévie, the Action de la Recherche Concertée, the Fonds Van Buuren, and the NATO International Scientific Exchange Programs.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.
§ Present address: Molecular Biology and Oncology, Dept. of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9148.
¶ The first two authors contributed equally to this work.
To whom correspondence should be addressed: National Institutes
of Health, Bldg. 5, Rm. 241, 5 Center Dr., Bethesda, MD 20892-0540.
2 M. Lobocka, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: GyrA, DNA gyrase A subunit; GyrB, DNA gyrase B subunit; GyrA59 and GyrA64, the 59- and 64-kDa N-terminal domain proteins of the DNA gyrase A subunit; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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