SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Broad-host-range plasmid RK2 encodes a post-segregational killing system, parDE, which contributes to the stable maintenance of this plasmid in Escherichia coli and many distantly related bacteria. The ParE protein is a toxin that inhibits cell growth, causes cell filamentation and eventually cell death. The ParD protein is a specific ParE antitoxin. In this work, the in vitro activities of these two proteins were examined. The ParE protein was found to inhibit DNA synthesis using an E. coli oriC supercoiled template and a replication-proficient E. coli extract. Moreover, ParE inhibited the early stages of both chromosomal and plasmid DNA replication, as measured by the DnaB helicase- and gyrase-dependent formation of FI*, a highly unwound form of supercoiled DNA. The presence of ParD prevented these inhibitory activities of ParE. We also observed that the addition of ParE to supercoiled DNA plus gyrase alone resulted in the formation of a cleavable gyrase–DNA complex that was converted to a linear DNA form upon addition of sodium dodecyl sulphate (SDS). Adding ParD before or after the addition of ParE prevented the formation of this cleavable complex. These results demonstrate that the target of ParE toxin activity in vitro is E. coli gyrase.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

A key feature of bacterial plasmids is their remarkably stable maintenance in growing populations of bacteria. This stability is the result of plasmid-encoded processes that ensure that there is a sufficient number of copies of a plasmid in a bacterial cell and that each daughter cell at cell division receives at least one copy of the plasmid element. Plasmids have been shown to encode a variety of regulatory mechanisms that sense plasmid copy number and that respond to upward or downward fluctuations in their copy number. It is also known that low- and moderate-copy-number plasmids are not randomly distributed to daughter cells on cell division but, instead, carry partitioning genes that ensure the distribution of plasmid copies to each daughter cell. The presence of post-segregational killing (PSK) systems on a plasmid also contributes to plasmid maintenance by virtue of the ability of the system to kill or inhibit the growth of a daughter cell that does not receive a copy of the plasmid during cell division.

Active PSK systems have now been described for a number of plasmids in a wide range of bacteria (Gerdes et al., 2000). One major class of PSK systems, designated proteic systems, consists of two proteins, one of which has toxin activity and the other has antitoxin activity by virtue of its ability to form a tight complex with the toxin protein. The best understood proteic PSK system is encoded by the F plasmid and consists of two proteins, CcdB, a toxin, and its antitoxin, CcdA (Karoui et al., 1983; Ogura and Hiraga, 1983; Miki et al., 1984; Jafféet al., 1985). E. coli cells that have lost the F plasmid are killed because the CcdB toxin is relatively stable, but the CcdA antitoxin is readily degraded by the Lon protease (Van Melderen et al., 1994). CcdB has been shown to kill E. coli by binding to the A subunit of DNA gyrase and inhibiting its activity (Bernard and Couturier, 1992; Bahassi et al., 1995). Plasmid R1 and the closely related plasmid R100 also carry a proteic PSK system that has been studied with respect to the mode of action of the toxin (Bravo et al., 1988; Tsuchimoto et al., 1988). The PSK system of these plasmids is identical and, in the case of plasmid R1, the toxin, Kid, has been shown to inhibit DnaB-dependent initiation of DNA replication (Ruiz-Echevarria et al., 1995).

Plasmid RK2 is a 60 kb broad-host-range plasmid that, despite the relatively low copy number of five to eight copies per chromosome, is stably maintained in a wide range of Gram-negative bacteria (Thomas and Helinski, 1989). One region of RK2 that is important in the stable maintenance of this plasmid is the 3.2 kb par region, which consists of five genes on two divergent operons, parCBA and parDE (Saurugger et al., 1986; Gerlitz et al., 1990; Roberts et al., 1990; Roberts and Helinski, 1992). Both operons have been shown to contribute to the stable maintenance of intact RK2 and mini-RK2 replicons in E. coli and in several Gram-negative bacteria distantly related to E. coli (Sia et al., 1995; Sobecky et al., 1996; Easter et al., 1997; 1998). ParA has been shown to be a resolvase, acting at an in cis site located between the two operons, which converts multimeric forms of the plasmid to monomers (Eberl et al., 1994). The functions of the ParB and ParC proteins are not known (Johnson et al., 1999). It is clear, however, that the 9 kDa ParD protein and the 12 kDa ParE protein function as a proteic PSK system, in which the antitoxin ParD protein protects the bacterial cell by forming a complex with the ParE toxin protein (Johnson et al., 1996). ParD exists as a dimer in solution and binds to the dimer form of the ParE toxin to form a tetrameric complex (Johnson et al., 1996; Oberer et al., 1999). Stabilization of mini-replicons of RK2 by parDE is accompanied by growth inhibition, filamentation of plasmid-free segregants and cell death (Roberts et al., 1994). In a comparison of the activities of the proteic PSK systems of plasmids F (ccd), R1 (parD) and RK2 (parDE), it was found that the parDE system was markedly more effective than the other two in stabilizing mini-R1 plasmids in E. coli (Jensen et al., 1995). In the present study, we demonstrate that purified ParE inhibits DNA replication in vitro and that this inhibition results from inactivation of E. coli DNA gyrase by the toxin. Furthermore, the inactivation of gyrase by ParE is prevented by the presence of the specific antitoxin, ParD and can be reversed by the addition of the antitoxin to ParE-inhibited gyrase.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

ParE inhibits E. coli oriC DNA replication in vitro

It is well established that the ParE protein is a toxin that causes extensive cell filamentation and that ParD is its specific antidote (Roberts et al., 1994; Johnson et al., 1996), but the mechanism of ParE action is not known. DAPI staining of ParE-induced cell filaments revealed a large nucleoid mass in the centre of the filaments (data not shown), suggesting that either DNA replication or segregation was inhibited. As interference with a number of processes that are important in maintaining chromosome structure and in chromosome partitioning, including DNA replication, is known to result in cell filamentation (Kornberg and Baker, 1992), we tested the effect of ParE on DNA replication in vitro. The assay used consisted of the Fraction II soluble protein extract of E. coli and an E. coli oriC plasmid DNA template. As shown in Fig. 1, ParE effectively inhibited DNA replication of oriC, and this inhibition, over a wide range of concentrations of the toxin, was prevented by the addition of the antitoxin, ParD.

image

Figure 1. ParE inhibits E. coli oriC DNA replication in vitro. Replication in vitro was performed using an E. coli C600 extract (Fraction II) active for oriC replication. Reactions containing 0.3 μg of supercoiled oriC DNA template and the indicated amounts of ParE protein were performed with and without the addition of 0.5 μg of ParD protein as indicated.

Download figure to PowerPoint

ParE inhibits F1* formation from a chromosomal and a plasmid replication origin

In an attempt to determine which stage of oriC DNA replication is inhibited by ParE, the effect of the toxin was examined on the formation of an extensively unwound form of covalently closed circular oriC DNA (FI*) (Baker et al., 1986). The formation of this electrophoretically distinct form of DNA is the net result of E. coli DnaA protein interaction at the origin of replication leading to the formation of an open complex followed by recruitment of the DnaB–DnaC complex to the origin region and DnaB helicase activation at this open region. The combined action of the DnaB helicase and E. coli gyrase in the presence of ATP generates this extensively unwound form. Nicking and closing of DNA by gyrase removes positive supercoils introduced by progression of the helicase complex on the DNA template (Gellert et al., 1976). As shown in Fig. 2A, ParE addition to the reaction mixture results in a signifi-cant reduction in the amount of FI* as well as a reduction in the levels of both supercoiled (FI) and open circular DNA (FIII) forms. Concomitantly, there is an increase in the amount of DNA that is at the electrophoretic position of the linear form (FII) of oriC DNA. The addition of ParD prevents ParE conversion of the various forms of oriC DNA to the linear form (Fig. 2A, lane 6). This effect of ParE in the FI* reaction is not specific for oriC DNA. Formation of the FI* form of the replication origin (oriV) of RK2 requires the RK2 replication initiation protein, TrfA, in addition to E. coli DnaA, DnaB and DnaC proteins and gyrase. A similar effect of ParE addition is seen with the supercoiled form of plasmid RK2 oriV DNA as the template (Fig. 2B). As with oriC DNA, ParE addition reduces the levels of the FI*, FI and FIII forms of oriV DNA and increases the level of the linear (FII) form. ParD once again neutralizes the ParE effect (Fig. 2B, lane 6). The observed inhibition of FI* formation could be a result of inhibition of helicase or gyrase activity or the inhibition of an earlier step in the formation of the prepriming complex at the replication origin.

image

Figure 2. ParE inhibits FI* formation from chromosomal and plasmid replication origins. Formation of FI*, the extensively unwound covalently closed circular DNA generated by helicase and gyrase activities, was tested as described in Experimental procedures using (A) E. coli chromosomal and (B) plasmid RK2 replication origins. For the initiation of oriC replication, the reactions contained the supercoiled form of template DNA, E. coli proteins DnaA, DnaB helicase, DNA gyrase and other components of the standard reaction mixture as described in Experimental procedures. For the initiation of RK2 oriV replication, the reactions contained supercoiled RK2 oriV template DNA, E. coli proteins DnaA, DnaB helicase, DnaC and DNA gyrase, the RK2-encoded TrfA initiation protein and other components of the standard reaction mixture. Increasing amounts of 100, 300 and 500 ng of ParE protein (lanes 3–5), respectively, or 500 ng of ParE protein together with 500 ng of ParD (lane 6) were added to the replication reactions. The positions of fast-migrating DNA (FI*), covalently closed circular DNA (FI), linear DNA (FII) and open circular DNA (FIII) are indicated.

Download figure to PowerPoint

A target of ParE inhibitory activity is E. coli gyrase

The conversion of oriC and oriV supercoiled DNA to the linear form by the addition of ParE to the FI* reaction mixture is reminiscent of the action of quinolone anti-biotics or the F plasmid CcdB toxin protein on gyrase (Maxwell, 1997; Couturier et al., 1998). In both cases, the covalently linked enzyme–DNA intermediate in the catalysis of negative supercoiling of DNA by gyrase is stabilized by these topoisomerase poisons and, upon addition of sodium dodecyl sulphate (SDS), usually added to stop the reaction and prepare the reaction mixture for agarose gel electrophoretic analysis, double-strand breakage of the DNA occurs. On the basis of these observations and the fact that gyrase is present in the FI* reaction mixture, ParE protein was tested for its effect on the activity of the E. coli gyrase enzyme. With the exception of gyrase, all the proteins that are required for FI* formation were omitted from the reaction mixture. As shown in Fig. 3A, ParE induced conversion of the supercoiled DNA form of the plasmid RK2 mini-replicon to the linear form (FII). Increasing the amount of gyrase results in increasing levels of the FII form of the DNA. At the highest concentration of gyrase, there is apparent fragmentation of the DNA to lower molecular weight forms. The presence of the antitoxin protein, ParD, in the reaction mixture prevents ParE-induced DNA breakage (Fig. 3B). The formation of the FII form of the DNA is dependent on the presence of ATP (Fig. 4A). In the absence of ATP and the presence of gyrase, there is relaxation of the supercoiled DNA, resulting in more slowly migrating forms (Fig. 4B). If ethidium bromide is present during the agarose electrophoretic separation of the various DNA forms, the various relaxed forms of plasmid DNA generated in the absence of ATP run as a single band (Fig. 4A). The observed conversion of the supercoiled DNA to a largely full-length linear form of DNA by ParE action on gyrase mimics the action of quinolones on this topoisomerase (Maxwell, 1997). As shown in Fig. 5, the quinolone, oxolinic acid, produces a single double-strand break in supercoiled DNA, and this product of oxolinic acid action is found at a position similar to that of ParE inhibition of gyrase. However, unlike what is observed with ParE, in which ParD prevents ParE-induced DNA breakage (Fig. 5, lane 3), ParD fails to neutralize the effect of oxolinic acid (Fig. 5, lane 5). This is consistent with the specific interaction between the ParD and ParE proteins.

image

Figure 3. A target of ParE inhibitory activity is E. coli gyrase. The reaction mixture for the gyrase assay described in Experimental procedures was identical to the standard reaction mixture with oriV supercoiled DNA template except that the DnaA, DnaB, DnaC, SSB, HU and TrfA proteins were omitted. DNA gyrase, ParE and ParD proteins were added to the reactions as indicated.

A. Increasing amounts of 125, 250, 500 and 1000 ng of gyrase without (lanes 2–5) and with 350 ng of ParE protein (lanes 6–9) were added to the reaction mixtures.

B. DNA gyrase (250 ng), ParE (350 ng) and ParD (1000 ng) were added to the reaction mixture as indicated. The positions of covalently closed circular DNA (FI), linear DNA (FII) and open circular DNA (FIII) are indicated.

Download figure to PowerPoint

image

Figure 4. ATP requirement for ParE inhibition of gyrase. The gyrase inhibition assay was performed as described in Experimental procedures. DNA gyrase (250 ng), ParE (350 ng) and ParD (1000 ng) proteins were added to reactions with and without ATP (2 mM) and an ATP regeneration system as indicated. After 30 min incubation at 32°C, reactions were terminated as described in Experimental procedures and analysed on a 1% TBE agarose gel run with (A) or without (B) ethidium bromide (0.1 μg ml–1). The positions of covalently closed circular DNA (FI), linear DNA (FII) and open circular DNA (FIII) are indicated.

Download figure to PowerPoint

image

Figure 5. Comparison of ParE and oxolinic acid inhibition of gyrase activity. The reaction mixtures were as described for the gyrase inhibition assay and were performed as described in Experimental procedures. ParE (300 ng), oxolinic acid (40 ng) and ParD (1000 ng) were added to the reaction mixture as indicated. Gyrase (160 ng) was present in all reactions. The positions of covalently closed circular DNA (FI), linear DNA (FII) and open circular DNA (FIII) are indicated.

Download figure to PowerPoint

Reversal of ParE inhibitory activity by ParD

To determine whether the effect of ParE on gyrase activity can be reversed by the antitoxin, ParD was added to the reaction mixture after the addition of the ParE protein. As shown in Fig. 6, the ParD protein is not only able to protect gyrase from the inhibitory action of ParE when both proteins are added before the addition of gyrase, but ParD is also able to reverse the formation in vitro of a ‘poisoned’ gyrase complex by ParE (Fig. 6). The addition of ParD for a 30 min period, after incubation of ParE with gyrase, ATP and supercoiled DNA for 30 min, completely reversed the formation of the cleavable gyrase–DNA complex (Fig. 6, lane 4). This result is similar to that found for the CcdA–CcdB post-segregational killing system of plasmid F, in which the action of the CcdB toxin protein in trapping gyrase in a cleavable complex can be reversed by the subsequent addition of CcdA antitoxin protein (Bahassi et al., 1999).

image

Figure 6. Reversal of ParE activity by ParD. The gyrase inhibition assay was performed as described in Experimental procedures except that two (I and II) sequential 30 min incubations were carried out at 32°C. After the second incubation, the reactions were terminated and analysed on a 1% TBE agarose gel. ParE (300 ng) and ParD (1000 ng) were added as indicated. Gyrase (160 ng) was present in all reactions. The positions of covalently closed circular DNA (FI), linear DNA (FII) and open circular DNA (FIII) are indicated.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The ParE toxin protein inhibits E. coli gyrase and converts supercoiled plasmid DNA to a singly cleaved linear form. By analogy with the extensive studies carried out on the mechanism of action of quinolone antibiotics (Maxwell, 1997) and the F plasmid CcdB protein (Couturier et al., 1998), it is likely that ParE interacts with one of the subunits of gyrase and stabilizes a gyrase–DNA cleavable complex. The addition of SDS, to stop the gyrase reaction before gel electrophoresis, results in the release of the double-strand DNA breaks in the ParE–gyrase–DNA complex and the generation of a linear form of the DNA. In view of the essential role of gyrase in DNA replication and the formation of a prepriming complex (F1*) on supercoiled DNA, it is not surprising that ParE is a strong inhibitor of these reactions in vitro (Figs 1 and 2). The activity of the ParD antitoxin in preventing this inhibition by ParE in vitro is consistent with the established activity of this antitoxin in vivo in preventing ParE killing (Roberts et al., 1994).

Despite the lack of amino acid sequence homology between ParE and the extensively studied CcdB protein (Roberts and Helinski, 1992), there are a number of properties that these two toxins share. Both proteins are relatively low-molecular-weight dimers in solution (Steyaert et al., 1993; Johnson et al., 1996). A linear DNA product of gyrase inhibition is generated by both ParE (Fig. 3) and CcdB (Bernard and Couturier, 1992) after treatment with SDS, not unlike that observed for the inhibitory activity of the quinolone oxolinic acid (Fig. 5) (Liu, 1989). However, unlike oxolinic acid, the inhibitory activity of both ParE (Fig. 4A) and CcdB (Bernard et al., 1993) requires ATP. Not surprisingly, given the specificity of the ParD protein in neutralizing the activity of ParE, the presence of this antitoxin fails to prevent oxolinic acid inactivation of gyrase (Fig. 5).

Escherichia coli gyrase is a type II topoisomerase and consists of two GyrA and two GyrB subunits (Reece and Maxwell, 1991; Maxwell, 1997). The GyrA subunits contain the catalytic core of the enzyme, whereas the GyrB subunits play a key role in ATP binding and hydrolysis. CcdB has been shown to target the GyrA subunit both in vitro and in vivo (Bernard and Couturier, 1992). Although there is no information to date as to which gyrase subunit is the target for ParE activity, it is known that an E. coli strain expressing the GyrA462 mutant protein, which is resistant to CcdB inhibition, is suscep-tible to killing by ParE (Roberts et al., 1994). Although most mutations to quinolone resistance are found in GyrA, several mutations have been mapped to the gyrB gene (Maxwell, 1997). The CcdB and quinolone-resistant mutations are located in different domains of GyrA, and the mutant proteins do not cross-protect (Couturier et al., 1998).

It is known for both quinolones and CcdB, and it is likely for ParE, that the mechanism of toxic activity is to trap gyrase into a cleavable complex. However, the subsequent events that lead ultimately to cell killing are not fully understood. The critical role of gyrase in maintaining the appropriate level of negative superhelical turns in DNA in vivo is well established, and interfering with this enzymatic activity is likely to lead to a cessation of cell growth. It has also been found that, in gyrA+/gyrA462 merodiploid strains, the CcdB-sensitive phenotype is dominant over the resistance phenotype (Bernard and Couturier, 1992). This observation and other evidence obtained on the mode of action of CcdB, quinolones and the antibiotic microcin B17 (Heddle et al., 2001) indicate that these agents both trap and poison the cleavable DNA–gyrase complex. The ‘poison’ hypothesis, in which the stabilized gyrase–inhibitor cleavable complex blocks the passage of polymerases along the DNA, has drawn support from the findings in vitro that the CcdB-stabilized complex blocks transcription by E. coli RNA polymerase (Critchlow et al., 1997). A quinolone-stabilized complex similarly serves as a barrier to DNA replication by T7 DNA polymerase (Wentzell and Maxwell, 2000). A block in replication fork progression could lead to the SOS response that is observed during CcdB killing of E. coli (Karoui et al., 1983; Sommer et al., 1985). It remains to be determined whether the cell killing activity of ParE results from the inhibition of gyrase-catalysed formation of negative supercoils or from the formation of a cleavable complex with gyrase that serves as a barrier to vital DNA processes and/or gives rise to DNA breaks. Until more information is available at the in vivo level on the activity of ParE, the mechanism(s) of ParE cytotoxicity remain(s) to be determined.

ParD effectively prevents the inactivation of gyrase by ParE when present at the time of addition of ParE or when added after incubation of ParE with gyrase and supercoiled DNA. In addition to its ability to form a tight tetrameric complex with ParE in the absence of DNA (Johnson et al., 1996), this antitoxin protein also regulates the expression of the parDE operon in E. coli (Davis et al., 1992). The ability of ParD to reverse the ParE stabilization of the gyrase cleavable complex (Fig. 6) suggests that ParD releases ParE from the toxin–gyrase complex, as has been shown for CcdA and CcdB (Bernard et al., 1993), and restores gyrase catalytic activity. If ParD is present in excess of ParE before the addition of gyrase in vitro, it is possible that the formation of a tight complex between the two Par proteins prevents binding of ParE to gyrase. The effectiveness of the parDE PSK system in the stable maintenance of RK2 and heterologous plasmids is presumably the result of pref-erential loss of active ParD in cells that have lost the plasmid resulting in a release of ParE activity. Unlike the anti-toxins CcdA and Kid, which are degraded by the ATP-dependent protease Lon in the absence of the corresponding toxin (Tsuchimoto et al., 1992; Van Melderen et al., 1994), a mutation in the lon gene in E. coli that inactivates the Lon protein failed to affect the ability of the parDE PSK system to stabilize a mini-RK2 plasmid (Roberts et al., 1994). This suggests that another protease or another mechanism is responsible for the presumed differential decay rate of the ParD antitoxin.

Several plasmid-encoded proteic systems have now been described, including ParD and ParE of plasmid RK2 (Roberts and Helinski, 1992), CcdB and CcdA of plasmid F (Ogura and Hiraga, 1983; Miki et al., 1984), Kis and Kid of plasmid R1 (identical to PemI and PemK of R100; Bravo et al., 1988; Tsuchimoto et al., 1988) and phd/doc of P1 (Lehnherr et al., 1993). Unlike the ParE and CcdB toxins that inactivate gyrase, the Kid protein of plasmid R1inhibits DnaB-dependent initiation of DNA replication in vitro (Ruiz-Echevarria et al., 1995). A non-proteic PSK system, designated hok/sok, is also encoded by the R1 plasmid (Gerdes et al., 1985; 2000). The expression of the Hok toxin protein results in the attenuation of cell growth and the appearance of ‘ghost-like’ cells, possibly as a result of disruption of the bacterial membrane by the Hok protein. Given the vast number of plasmid elements in the wide range of bacteria residing in mammalian, terrestrial and aquatic environments and the likelihood that a high proportion of these plasmids encode a post-segregational killing system, it will not be surprising that the toxin proteins expressed by these systems bring about bacterial cell growth arrest or killing by a variety of mechanisms. In addition, the effectiveness of a PSK system undoubtedly depends upon the host cell in which the toxin is expressed. In comparative studies in which the parDE system, carried by plasmid RK2, was tested for its contribution to plasmid maintenance in different E. coli strains and five bacteria distantly related to E. coli, it was found that the effectiveness of the parDE system in promoting stable maintenance of the plasmid varied with the bacterial host (Sia et al., 1995; Easter et al., 1997; 1998). In another study, the RK2 parDE system was found to be considerably more effective in the maintenance of mini-R1 plasmids in E. coli than the F plasmid CcdA/CcdB system (Jensen et al., 1995). These host and PSK system differences in the stable maintenance of a plasmid are likely to result from a number of factors including differences in the expression of a PSK system in different hosts, internal environmental effects on the toxin– antitoxin interaction, differences in host chaperone activity, variation in antitoxin inactivation and differences in the sensitivity of the target in different bacteria. Stabilization of plasmid maintenance by parD/parE is of particular interest because these genes are on a plasmid that is stably maintained in a wide range of Gram-negative bacteria and the fact that this proteic system has been shown to be active in a number of distantly related bacteria. It is therefore of particular interest to confirm that the in vivo target of the ParE toxin is indeed gyrase and to understand the unique properties of this toxin that allow it to function in such a wide range of bacterial hosts.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains, proteins, plasmids, and reagents

Escherichia coli C600 (thr, leu, thi, supE44, tonA) (Appleyard, 1954) was used in this study for the preparation of a crude extract (Fraction II) active in DNA replication. The RK2-encoded proteins ParE and ParD, purified as described previously (Roberts et al., 1993; Johnson et al., 1996), were generously provided by Dr E. Johnson. The His6-tagged mutant TrfA33 (254D/267L) version of the TrfA mutant protein, which contains two plasmid copy-up mutations and is fully functional in vivo and in vitro (Konieczny and Helinski, 1997a), was purified as described previously (Blasina et al., 1996). Protocols and bacterial strains used for the purification of E. coli gyrase were kindly provided by Dr N. Dixon (Australian National University). Purification of E. coli DnaC and the histidine-tagged versions of E. coli DnaA and DnaB proteins was carried out as described previously (Li and Crooke, 1999; Caspi et al., 2001). HU protein was a kind gift from Dr R. McMacken. pKD19L1 carrying the minimal RK2 origin region and plasmid pBSoriC carrying the oriC DNA origin fragment have been described previously (Yung and Kornberg, 1989; Doran et al., 1998). pYJ2 is an oriC plasmid constructed by the insertion of an EcoRI fragment containing the RK2 tetAR genes into pBSoriC. The E. coli SSB proteins were obtained from Promega, creatine kinase, bovine serum albumin (Fraction V), creatine phosphate and rNTPs from Sigma, dNTPs from Pharmacia, and [3H-methyl]-dTTP from ICN Radiochemicals.

Escherichia coli oriC DNA replication using crude extracts

Preparation of the E. coli crude Fraction II extract and reaction conditions was carried out essentially as described previously (Kittell and Helinski, 1991). The standard reactions contained 300 ng of pBSoriC template. Incubation was at 32°C for 60 min. Reactions were stopped by the addition of trichloroacetic acid, and total nucleotide incorporation (pmol) into DNA was measured by liquid scintillation counting, after filtration of the reaction mixture onto Whatman GF/C glassfibre filters.

Template unwinding assay (FI*)

Helicase unwinding assays (FI* formation) were performed as described previously (Konieczny and Helinski, 1997b). The plasmid pYJ2 (300 ng) containing the E. coli chromo-somal origin or plasmid pKD19L1 (300 ng) containing the RK2 replication origin were used as DNA templates. Unless noted otherwise, proteins were used in the following amounts: DnaA (320 ng), DnaB (600 ng), DnaC (120 ng), HU (5 ng), gyrase (120 ng) and SSB (230 ng). The oriV-dependent reaction was supplemented with RK2 replication initiation protein TrfA 254D/267L (500 ng). The indicated amounts of ParE and ParD proteins were added to the reaction mixtures. Reactions (25 μl) were assembled on ice in buffer containing 40 mM HEPES/KOH, pH 8.0, 25 mM Tris-HCl, pH 7.4, 80 μg ml–1 BSA, 4% sucrose; 4 mM dithiothreitol (DTT), 11 mM magnesium acetate, 2 mM ATP, 8 mM creatine phosphate and 20 μg ml–1 creatine kinase, and then incubations were carried out at 32°C for 30 min. After incubation, the reactions were stopped by the addition of 10 mM EDTA and 2% SDS (final concentration), followed by 2 min incubation at 65°C. Aliquots of 10% sucrose and 0.05% bromophenol blue (final concentration) were then added to the reaction mixture. The mixture was analysed on a 1% agarose gel in TBE buffer (0.09 M Tris-borate, 0.002 M EDTA). The samples were electrophoresed at 25 V for 20 h, and the gel was stained with ethidium bromide solution.

Gyrase inhibition assay

The standard reaction (25 μl) contained supercoiled pKDL19 DNA (300 ng), the indicated amounts of E. coli gyrase and, unless noted otherwise, ParE (350 ng), ParD (1000 ng) or oxolinic acid (40 ng). The reactions were carried out in the same buffer as that used for the template unwinding assay and incubated, terminated and analysed as described for the FI* assay.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr Nick Dixon for bacterial strains and protocols for the purification of the gyrase enzyme, and Dr Aresa Toukdarian for critical reading of this manuscript. This work was supported by a National Institutes of Health Research Grant AI-07194 awarded to D.R.H.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Appleyard, R.K. (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39: 440452.
  • Bahassi, E.M., O’Dea, M.H., Allali, N., Messens, J., Gellert, M., and Couturier, M. (1999) Interactions of CcdB with DNA gyrase. Inactivation of GyrA, poisoning of the gyrase-DNA complex & the antidote action of CcdA. J Biol Chem 274: 1093610944.
  • Bahassi, E.M., Salmon, M.A., Vanmelderen, L., Bernard, P., and Couturier, M. (1995) F plasmid CcdB killer protein – CcdB gene mutants coding for non-cytotoxic proteins which retain their regulatory functions. Mol Microbiol 15: 10311037.
  • Baker, T.A., Sekimizu, K., Funnell, B.E., and Kornberg, A. (1986) Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell 45: 5364.
  • Bernard, P., and Couturier, M. (1992) Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J Mol Biol 226: 735745.
  • Bernard, P., Kézdy, K.E., Van Melderen, L., Steyaert, J., Wyns, L., Pato, M.L., et al. (1993) The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. J Mol Biol 234: 534541.
  • Blasina, A., Kittell, B.L., Toukdarian, A.E., and Helinski, D.R. (1996) Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins. Proc Natl Acad Sci USA 93: 35593564.
  • Bravo, A., Ortega, S., De Torrontegui, G., and Díaz, R. (1988) Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1. Mol Gen Genet 215: 146151.
  • Caspi, R., Pacek, M., Consiglieri, G., Helinski, D.R., Toukdarian, A., and Konieczny, I. (2001) A broad host range replicon with different requirements for replication initiation in three bacterial species. EMBO J 20: 32623271.
  • Couturier, M., Bahassi, E.M., and Van Melderen, L. (1998) Bacterial death by DNA gyrase poisoning. Trends Microbiol 6: 269275.
  • Critchlow, S.E., Odea, M.H., Howells, A.J., Couturier, M., Gellert, M., and Maxwell, A. (1997) The interaction of the F plasmid killer protein, CcdB, with DNA gyrase: Induction of DNA cleavage and blocking of transcription. J Mol Biol 273: 826839.DOI: 10.1006/jmbi.1997.1357
  • Davis, T.L., Helinski, D.R., and Roberts, R.C. (1992) Transcription and autoregulation of the stabilizing functions of broad-host-range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa. Mol Microbiol 6: 19811994.
  • Doran, K.S., Konieczny, I., and Helinski, D.R. (1998) Replication origin of the broad host range plasmid RK2: positioning of various motifs is critical for initiation of replication. J Biol Chem 273: 84478453.
  • Easter, C.L., Sobecky, P.A., and Helinski, D.R. (1997) Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2. J Bacteriol 179: 64726479.
  • Easter, C.L., Schwab, H., and Helinski, D.R. (1998) Role of the parCBA operon of the broad-host-range plasmid RK2 in stable plasmid maintenance. J Bacteriol 180: 6023 6030.
  • Eberl, L., Kristensen, C.S., Givskov, M., Grohmann, E., Gerlitz, M., and Schwab, H. (1994) Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4. Mol Microbiol 12: 131 141.
  • Gellert, M., Mizuuchi, K., O’Dea, M.H., and Nash, H.A. (1976) DNA gyrase: an enzyme that introduces superhelical turns into DNA.. Proc Natl Acad Sci USA 73: 38723876.
  • Gerdes, K., Larsen, J.E., and Molin, S. (1985) Stable inheritance of plasmid R1 requires two different loci. J Bacteriol 161: 292298.
  • Gerdes, K., Ayora, S., Canosa, I., Ceglowski, P., Díaz-Orejas, R., Franch, T., et al. (2000) Plasmid maintenance systems. In The Horizontal Gene Pool. Thomas, C.M. (eds). Amsterdam: Harwood Academic Publishers, pp. 4985.
  • Gerlitz, M., Hrabak, O., and Schwab, H. (1990) Partitioning of broad-host-range plasmid RP4 is a complex system involving site-specific recombination. J Bacteriol 172: 61946203.
  • Heddle, J.G., Blance, S.J., Zamble, D.B., Hollfelder, F., Miller, D.A., Wentzell, L.M., et al. (2001) The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition. J Mol Biol 307: 12231234.
  • Jaffé, A., Ogura, T., and Hiraga, S. (1985) Effects of the ccd function of the F plasmid on bacterial growth. J Bacteriol 163: 841849.
  • Jensen, R.B., Grohmann, E., Schwab, H., Diaz-Orejas, R., and Gerdes, K. (1995) Comparison of ccd of F, parDE of RP4 & parD of R1 using a novel conditional replication control system of plasmid R1. Mol Microbiol 17: 211 220.
  • Johnson, E.P., Strom, A.R., and Helinski, D.R. (1996) Plasmid RK2 toxin protein ParE: purification and inter-action with the ParD antitoxin protein. J Bacteriol 178: 14201429.
  • Johnson, E.P., Mincer, T., Schwab, H., Burgin, A.B., and Helinski, D.R. (1999) Plasmid RK2 ParB protein: Purification and nuclease properties. J Bacteriol 181: 60106018.
  • Karoui, H., Bex, F., Drèze, P., and Couturier, M. (1983) Ham22, a mini-F mutation which is lethal to host cell and promotes recA-dependent induction of lambdoid prophage. EMBO J 2: 18631868.
  • Kittell, B.L., and Helinski, D.R. (1991) Iteron inhibition of plasmid RK2 replication in vitro: evidence for intermolecular coupling of replication origins as a mechanism for RK2 replication control. Proc Natl Acad Sci USA 88: 13891393.
  • Konieczny, I., and Helinski, D.R. (1997a) The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone. Proc Natl Acad Sci USA 94: 1437814382.
  • Konieczny, I., and Helinski, D.R. (1997b) Helicase delivery and activation by DnaA and TrfA proteins during the initiation of replication of the broad host range plasmid RK2. J Biol Chem 272: 3331233318.
  • Kornberg, A., and Baker, T.A. (1992) DNA Replication. Freeman Press, New York.
  • Lehnherr, H., Maguin, E., Jafri, S., and Yarmolinsky, M.B. (1993) Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage & phd, which prevents host death when prophage is retained. J Mol Biol 233: 414428.DOI: 10.1006/jmbi.1993.1521
  • Li, Z., and Crooke, E. (1999) Functional analysis of affinity-purified polyhistidine-tagged DnaA protein. Protein Expr Purif 17: 4148.
  • Liu, L.F. (1989) DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 58: 351375.
  • Maxwell, A. (1997) DNA gyrase as a drug target. Trends Microbiol 5: 102109.
  • Miki, T., Chang, Z.T., and Horiuchi, T. (1984) Control of cell division by sex factor F in Escherichia coli. II. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment. J Mol Biol 174: 627646.
  • Oberer, M., Lindner, H., Glatter, O., Kratky, C., and Keller, W. (1999) Thermodynamic properties and DNA binding of the ParD protein from the broad host-range plasmid RK2/RP4 killing system. Biol Chem 380: 14131420.
  • Ogura, T., and Hiraga, S. (1983) Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci USA 80: 47844788.
  • Reece, R.J., and Maxwell, A. (1991) DNA gyrase: structure and function. Crit Rev Biochem Mol Biol 26: 335375.
  • Roberts, R.C., and Helinski, D.R. (1992) Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J Bacteriol 174: 81198132.
  • Roberts, R.C., Burioni, R., and Helinski, D.R. (1990) Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J Bacteriol 172: 6204 6216.
  • Roberts, R.C., Spangler, C., and Helinski, D.R. (1993) Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2. J Biol Chem 268: 27109 27117.
  • Roberts, R.C., Ström, A.R., and Helinski, D.R. (1994) The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss. J Mol Biol 237: 3551.DOI: 10.1006/jmbi.1994.1207
  • Ruiz-Echevarria, M.J., Gimenez-Gallego, G., Sabariegos-Jareno, R., and Diaz-Orejas, R. (1995) Kid, a small protein of the parD stability system of plasmid R1, is an inhibitor of DNA replication acting at the initiation of DNA synthesis. J Mol Biol 247: 568577.
  • Saurugger, P.N., Hrabak, O., Schwab, H., and Lafferty, R.M. (1986) Mapping and cloning of the par-region of broad-host-range plasmid RP4. J Biotechnol 4: 333343.
  • Sia, E.A., Roberts, R.C., Easter, C., Helinski, D.R., and Figurski, D.H. (1995) Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol 177: 27892797.
  • Sobecky, P.A., Easter, C.L., Bear, P.D., and Helinski, D.R. (1996) Characterization of the stable maintenance pro-perties of the par region of broad-Host-range plasmid RK2. J Bacteriol 178: 20862093.
  • Sommer, S., Bailone, A., and Devoret, R. (1985) SOS induction by thermosensitive replication mutants of miniF plasmid. Mol Gen Genet 198: 456464.
  • Steyaert, J., Van Melderen, L., Bernard, P., Thi, M.H., Loris, R., Wyns, L., and Couturier, M. (1993) Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein. J Mol Biol 231: 513515.DOI: 10.1006/jmbi.1993.1301
  • Thomas, C.M., and Helinski, D.R. (1989) Vegetative replication and stable inheritance of IncP plasmids. In Promiscuous Plasmids of Gram-Negative Bacteria. Thomas, C.M. (eds). San Diego: Academic Press, pp. 125.
  • Tsuchimoto, S., Ohtsubo, H., and Ohtsubo, E. (1988) The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth. J Bacteriol 170: 14611466.
  • Tsuchimoto, S., Nishimura, Y., and Ohtsubo, E. (1992) Two genes, pemK and pemI, responsible for stable maintenance of resistance plasmid R100. J Bacteriol 174: 42054211.
  • Van Melderen, L., Bernard, P., and Couturier, M. (1994) Lon-dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid-free segregant bacteria. Mol Microbiol 11: 11511157.
  • Wentzell, L.M., and Maxwell, A. (2000) The complex of DNA gyrase and quinolone drugs on DNA forms a barrier to the T7 DNA polymerase replication complex. J Mol Biol 304: 779791.DOI: 10.1006/jmbi.2000.4266
  • Yung, B.Y.M., and Kornberg, A. (1989) The DnaA initiator protein binds separate domains in the replication origin of Escherichia coli. J Biol Chem 264: 61466150.