Interactions of CcdB with DNA Gyrase
INACTIVATION OF GyrA, POISONING OF THE GYRASE-DNA COMPLEX, AND THE ANTIDOTE ACTION OF CcdA*

El Mustapha BahassiDagger §, Mary H. O'Deaparallel , Noureddine AllaliDagger , Joris Messens**, Martin Gellertparallel Dagger Dagger , and Martine CouturierDagger

From the Dagger  Laboratoire de Génétique des Procaryotes, Département de Biologie Moléculaire, Université Libre de Bruxelles, rue des Chevaux 67, B-1640 Rhode-Saint-Genèse, Belgium, the parallel  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains and Plasmids-- MAX Efficiency DH5alpha 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.

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-beta -D-galactopyranoside induction in DH5alpha 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.

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. DH5alpha F'IQTM, which also produces the LacIq repressor, was used as the host strain.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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.

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).


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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.

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).


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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.

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.


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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.

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).


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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.

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.


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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

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-beta -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.

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger Dagger 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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