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Antimicrob Agents Chemother. Mar 2003; 47(3): 941–947.
PMCID: PMC149342

Alteration of Escherichia coli Topoisomerase IV to Novobiocin Resistance

Abstract

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.

Coumarins and quinolones are two important classes of antimicrobial agents that share as their targets bacterial type II topoisomerases (20). The two Escherichia coli type II topoisomerases, DNA gyrase and topoisomerase IV (topo IV), are necessary for cell viability, playing vital roles in DNA replication, chromosome segregation, and DNA compaction (28). By actively introducing negative supercoils into DNA, gyrase removes the positive supercoils caused by DNA replication and helps to maintain the steady-state balance of negative supercoiling of the bacterial DNA (44). Topo IV's primary role is in the decatenation of daughter chromosomes following DNA replication (22, 35, 48), but it also contributes to the advance of the replication fork by removing positive supercoils (24). Both enzymes carry out their functions by coupling the energy of ATP hydrolysis to the directional passage of one double-strand of DNA through another (14, 39). The enzymatic labor is divided between the two subunits of each enzyme: GyrA and GyrB for gyrase and ParC and ParE for topo IV. The GyrB and ParE subunits contain nucleotide binding sites, whereas the GyrA and ParC subunits are responsible for DNA breakage and reunion. Gyrase and topo IV are heterotetramers in the forms of A2B2 and C2E2, respectively.

The quinolones were first shown to inhibit DNA gyrase (13, 42) and subsequently were demonstrated to inhibit topo IV (23, 25, 35). The coumarins have long been known to inhibit gyrase (15). Coumarins and quinolones inhibit topoisomerases by starkly different mechanisms. The coumarin antibiotics, such as novobiocin, are competitive inhibitors of ATP and bind in a pocket overlapping the ATP binding site of GyrB (29, 41). Quinolones, instead, kill cells by a dominant poison mechanism, through the breakage of DNA (4, 19, 26). Quinolone-resistant E. coli strains contain mutations in one or both genes encoding the subunits of gyrase (46, 47) and of topo IV (1, 18, 27, 43). Because gyrase is the primary target of quinolones for E. coli, a strain must first have a mutation rendering gyrase resistant in order to acquire a subsequent mutation in the topo IV genes (1, 10). Most often the protein alterations are in GyrA and ParC. Interestingly, in some gram-positive organisms, the hierarchy is reversed and topo IV is the primary target (12, 34).

Heretofore, alterations of topoisomerases to confer resistance to coumarin antibiotics have affected only GyrB (32). Several lines of evidence have suggested, though, that topo IV might be a secondary target of novobiocin in vivo. First, novobiocin inhibits the activity of purified topo IV, albeit inefficiently. The Ki of novobiocin for purified topo IV is 1 to 2 orders of magnitude higher than that for purified gyrase (15, 35, 41). Second, whereas low concentrations of novobiocin inhibit only gyrase, leading to a slow stop of DNA replication, high concentrations of novobiocin cause a fast stop of replication, indicative of a secondary target (24). Topo IV was implicated as the probable secondary target because catenated plasmids accumulated at high novobiocin concentrations. Third, the structure of the ParE active site is strikingly similar to that for GyrB (45; D. Wigley, personal communication).

We sought to isolate a mutant with a topo IV resistant to novobiocin for three reasons. First, the definitive proof that topo IV is a secondary target of coumarins requires demonstration that an alteration in the protein confers resistance in vitro and in vivo. Second, the set of single- and double-mutant strains that confer resistance of gyrase and/or topo IV to quinolones has been instrumental for decades in elucidating the intricate physiological roles of topoisomerases (13, 17, 49). Because quinolones rapidly lead to double-strand breaks of DNA trapped by covalently bound topoisomerases, secondary consequences, such as SOS induction, follow drug administration that can, however, complicate interpretation of results (16, 21). Coumarins do not show such confounding effects, but the essential strains needed to ensure that the drugs have a single target were not available. Finally, we reasoned that a resistant mutant might show how coumarins inhibit topo IV and demonstrate a path that resistance to these drugs takes.

We show that an alteration in ParE near the ATP binding cleft confers high-level novobiocin resistance to topo IV and blocks the action of the drug on a characteristic activity of topo IV in vivo.

MATERIALS AND METHODS

Strain construction.

The codon for arginine 132 of the parE gene was chosen for mutagenesis because Arg-136 in the equivalent position of GyrB is frequently altered in novobiocin-resistant gyrase isolates (see Results). We changed the parE Arg-132 codon (CGC) to encode Cys (TGC), Ser (AGC), and His (CAC) in anticipation that each mutation might have a different effect on the growth rate and/or drug resistance. The mutations were constructed and inserted into the chromosomes of the desired strains (Table (Table1)1) by the following method. parE and the 5′ upstream region were individually amplified from chromosomal DNA by PCR using primers with restriction sites engineered at the 5′ ends. The chloramphenicol resistance gene, cat, of pACYC184 (New England Biolabs) was amplified using the same strategy, and the three fragments were sequentially ligated into pBluescript (Stratagene). This plasmid was subjected to site-directed mutagenesis using complementary mismatched primers and PCR-based linear amplification of the DNA with Vent polymerase (New England Biolabs), followed by removal of parental plasmid by restriction with DpnI (New England Biolabs). DNA sequencing confirmed that the Arg-132 codon had been changed to that encoding Cys, Ser, or His. The upstream parE-cat-parE cassettes were then amplified, and the linear PCR products were used to transform recD cells made competent by CaCl2 treatment (37). Integration of the cassette was selected by chloramphenicol resistance. The CGC(Arg)→TGC(Cys) mutation at codon 132 (R132C) was confirmed by nucleotide sequencing of the chromosomal insert. Integration into the chromosome was also achieved for the serine mutation but was lost after several generations of growth, suggesting instability of this point mutation. No transformants were obtained using the His cassette.

TABLE 1.
E. coli strains useda

The parER132C mutation was transduced by P1 phage into a gyrB234 (coumermycin resistant, temperature sensitive) strain and its isogenic gyrB+ strain. Novobiocin is actively pumped out of E. coli by the AcrAB multidrug efflux pump (31). Increased drug sensitivity was attained by introduction of the acrA::kan allele by P1 transduction. A summary of the strains used in this work is presented in Table Table11.

Purification of the ParE R132C protein.

The expression vector for the ParE protein, pET11-parE (obtained from H. Hiasa), was subjected to PCR-based linear amplification using complementary mismatched primers as described above. The plasmid DNA was sequenced to confirm that the codon for Arg-132 had been changed to encode cysteine. The plasmid was then transformed into E. coli BL21(DE3) (40) to direct the isopropyl-β-d-thiogalactopyranoside-inducible expression of the ParE R132C protein. The altered protein was induced and purified according to Peng and Marians (36), except that fraction 2 was diluted to 45 mM NaCl immediately before loading onto a DE-52 column, which had been equilibrated in buffer A + 45 mM NaCl. The peak fractions from the DE-52 column were pooled and dialyzed against topo IV storage buffer for use in activity assays. The protein was estimated to be 90% pure by densitometer analysis of a Coomassie blue-stained polyacrylamide gel. Wild-type ParC and ParE were purified to near-homogeneity using the purification scheme described above followed by a final Superdex 200 column.

Topo IV activity assays.

The measurement of the relaxation of negative supercoils by topo IV was carried out as described previously (7). pUC18, 2.7 kb, was used as a substrate. The products were analyzed by gel electrophoresis through a 1.2% agarose gel in Tris-acetate-EDTA.

Positive supercoiling assays.

Strains 2817 (gyrB+ parE+ acr), 2818 (gyrB+ parER132C acr), 2834 (gyrB234 parE+ acr), and 2832 (gyrB234 parER132C acr) were transformed with pBR322 (4.4 kb) using the Transformaid kit (Fermentas). Cultures were grown to log phase in Luria broth (LB) plus 15 μg of tetracycline (Sigma)/ml and treated with the indicated concentrations of novobiocin (Sigma) for 20 min with shaking. All experiments were carried out at 30°C because of the temperature sensitivity conferred by the gyrB234 allele. Plasmid DNA from 2 ml of culture was prepared using miniprep columns (Qiagen), and the DNA was resolved by electrophoresis through a 1% agarose gel containing 5 μg of chloroquine (Sigma)/ml in Tris-acetate-EDTA running buffer. The relaxed DNA marker was produced by treatment of pBR322 with wheat germ topoisomerase I. Topoisomers were visualized by Southern blotting using probe generated with the Ready-To-Go DNA labeling kit (Amersham Pharmacia).

Plating assays.

The four acr strains of interest (2817, 2818, 2834, and 2832) were grown in LB plus 50 μg of kanamycin (Sigma)/ml to an optical density of 600 nm (OD600) of 0.1 to 0.4. The cultures were then diluted 105-fold and plated in duplicate onto LB plates containing 0, 2.5, 5, 7.5, 10, and 15 μg of novobiocin/ml for the strains with wild-type gyrase. LB plates containing 0 to 50 μg of novobiocin/ml, in 10-μg/ml increments, were used for the resistant gyrase strains. The plates were incubated at 30°C for 2 to 4 days, after which colonies were counted. The experiment was repeated exactly using independent cultures of the four strains.

The two gyrB+ strains have very similar doubling times in LB. The two gyrB234 strains also grow at the same rates, as monitored by light absorbance. However, the four strains differ significantly in the number of viable cells per OD600. Compared to the gyrB+ parE+ strain, the gyrB234 parE+ strain has around 75% as many viable cells per OD600; the gyrB+ parE+ strain has approximately 55% of the number of viable cells; and the gyrB234 parER132C strain has only 18% the number of viable cells per OD600. These observations suggest that the double mutant undergoes severe filamentation and/or produces a significant number of inviable cells, possibly indicating a partition defect phenotype.

RESULTS

Design of the parER132C mutant strains.

Because of the structural similarities between gyrase and topo IV, we hypothesized that novobiocin inhibits topo IV in a manner similar to that for gyrase, by blocking the binding of ATP to the ParE subunit of topo IV. In choosing an amino acid residue to target for obtaining a drug-resistant mutant, we took advantage of the high degree of sequence homology between GyrB and ParE and the good overlay between their respective crystal structures (45; D. Wigley, personal communication). GyrB and ParE have 47% sequence identity and 66% sequence similarity over the first 220 amino acids (Fig. (Fig.1A).1A). GyrB Arg-136 is the most commonly altered residue in novobiocin-resistant E. coli isolates (6). This arginine hydrogen bonds to the coumarin ring of novobiocin in the crystal structure of the GyrB N-terminal 24-kDa fragment complexed with novobiocin (29). The residue is conserved between GyrB and ParE (Fig. (Fig.1A)1A) and occupies a similar position near the ATP binding cleft in the GyrB and ParE crystal structures (Fig. (Fig.1B)1B) (D. Wigley, personal communication). Accordingly, ParE Arg-132 was changed to cysteine; the mutation is referred to hereafter as parER132C.

FIG. 1.
(A) Alignment of the ParE and GyrB N-terminal amino acid sequences. Amino acids listed between the ParE and GyrB lines indicate sequence identity, and plus signs indicate amino acid similarity. Residues known to be important for binding of ATP to GyrB ...

The AcrAB outer membrane transport system actively pumps novobiocin out of cells (31). We therefore knocked out the acrA gene to make the set of strains tested in this work approximately 25 times more susceptible to novobiocin (data not shown).

The ParE R132C protein is highly resistant to inhibition by novobiocin and is less active than wild-type ParE.

To provide a quantitative measure of the level of resistance gained by alteration of ParE Arg-132 to cysteine, we purified the ParE R132C protein and assayed its activity relative to that of wild-type ParE. Figure Figure2A2A shows a titration of the wild-type ParE protein in the presence of an excess of ParC. Relaxation of the negatively supercoiled substrate required 4 ng of wild-type ParE. In comparison, 18 ng of ParE R132C was required to relax a negatively supercoiled plasmid to the same extent (Fig. (Fig.2B).2B). Thus, ParE R132C is 22% as active as the wild-type protein. There is no contaminating ParC or topoisomerase activity in the wild-type or altered ParE preparations, since omission of ParC from the assays abolished all relaxation activity (Fig. (Fig.2A2A and B, rightmost lanes). When equal amounts of wild-type ParE and ParE R132C were mixed in the presence of excess ParC, the wild-type protein retained full activity, indicating that no diffusible inhibitor is present in the ParE R132C preparation (Fig. (Fig.2C).2C). The reduced activity of the ParE R132C protein is similar to the equivalently altered GyrB R136C protein, which has 13% of the wild-type supercoiling activity (6). The diminution of activity is expected because of the close association of the alterations to the ATP binding sites of each enzyme.

FIG. 2.
Wild-type and altered topo IV activity assays in vitro. Relaxed (Rel) and negatively supercoiled [(-) sc] DNAs are indicated. (A) Titration of wild-type ParE. The indicated amount of wild-type ParE protein was incubated with 150 ng of negatively supercoiled ...

To quantify the level of resistance conferred by the ParE R132C alteration, we titrated novobiocin into activity assays containing either wild-type ParE or ParE R132C. Approximately 40 times more novobiocin was required to inhibit topo IV activity in the ParE R132C reactions (40 μg/ml for ParE R132C versus 1 μg/ml for wild-type ParE) (Fig. (Fig.2D).2D). We conclude that novobiocin inhibits topo IV by the same mechanism by which it inhibits DNA gyrase, since alteration of homologous amino acids confers substantial resistance onto both enzymes.

The ParE R132C alteration confers novobiocin resistance to topo IV in vivo as assayed by positive supercoil relaxation.

Topo IV's recently appreciated penchant for removing positive supercoils (7) enabled us to assay selectively topo IV activity in vivo. High concentrations of novobiocin had been shown to hit a secondary target in addition to gyrase (24). If that target is topo IV, then the ParE R132C alteration should allow topo IV to relax a positively supercoiled plasmid in vivo at high concentrations of novobiocin. Positive supercoils are efficiently generated in a plasmid by the twin domain effect (30). Active transcription of genes anchored within the cell, such as the tet gene of pBR322, generates positive supercoils ahead of and negative supercoils behind a vigorously transcribing RNA polymerase. In a wild-type cell, where all the relevant topoisomerases are active, the plasmid will remain negatively supercoiled during transcription. If gyrase alone is inhibited, the DNA will be relaxed by the action of other topoisomerases. But if both gyrase and topo IV are inhibited, then the DNA will be positively supercoiled.

parE+ and parER132C strains were assayed for their ability to relax positive supercoils in the presence of novobiocin in both gyrB+ and gyrB234 backgrounds (Fig. (Fig.3).3). The effects of novobiocin on the supercoiling of pBR322 were visualized by agarose gel electrophoresis in the presence of the DNA intercalator, chloroquine. Chloroquine introduces positive supercoils into DNA. The amount used in these gels removes enough negative supercoils in native pBR322 to resolve its constituent topoisomers, which are still negatively supercoiled (Fig. (Fig.3A3A and B, lanes 1 and 6). Relaxed pBR322 becomes positively supercoiled (lanes labeled “Rel”), and a positively supercoiled plasmid would become more highly positively supercoiled and migrate faster. As increasing concentrations of novobiocin were added to the parE+ strains, a significant percentage of the plasmid ran as positively supercoiled (Fig. (Fig.3A3A and B, lanes 3 to 5), as anticipated from the inhibition of both gyrase and topo IV. For the parER132C strains, accumulation of positive supercoils required much higher concentrations of novobiocin, indicating that the altered topo IV is resistant over most of the concentration range tested (lanes 7 to 9).

FIG. 3.
Positive supercoiling assay for topo IV activity in vivo. (A and B) Gel electrophoresis of intrinsic pBR322 in the four acr strains indicated (gyrBr represents gyrB234). Plasmid was isolated from each strain and run on a 1% agarose gel containing 5 μg ...

We draw two conclusions. First, the parER132C mutation confers upon topo IV an approximately 20-fold increase in resistance to novobiocin, as measured by the concentration of novobiocin needed to induce an equivalent level of positive supercoiling (Fig. (Fig.3A,3A, lanes 3 and 10). Therefore, topo IV is indeed a secondary target of novobiocin to gyrase in vivo. Second, while the effect of the gyrase resistance allele is readily apparent at low novobiocin concentrations (compare lanes 2 in Fig. Fig.3A3A and B), at higher concentrations of novobiocin, positive supercoils accumulate to similar extents in both the gyrB+ and gyrB resistant mutant backgrounds (Fig. (Fig.3A3A and B, lanes 3 to 5). For DNA to be positively supercoiled, both gyrase and topo IV must be inhibited by novobiocin. Therefore, much of the resistance of the gyrase mutant is overcome before topo IV is inhibited.

The parER132C mutation confers a modest increase in viability in the presence of novobiocin.

We next measured the influence of the parER132C mutation on viability in the presence of novobiocin. The data in Fig. Fig.44 summarize the results of two experiments in which strains containing each combination of gyrB+/gyrB234 and parE+/parER132C alleles were plated onto increasing concentrations of novobiocin. The results were as expected. A single mutation to novobiocin resistance in gyrase, but not one in topo IV, led to greatly enhanced viability in the presence of novobiocin (Fig. (Fig.4).4). The double mutant in which topo IV and gyrase are both resistant showed only a small advantage in growth compared to the gyrB234 strain (Fig. (Fig.4).4). Both resistant gyrase strains required at least five times more novobiocin to get a reduction in viability comparable to that of the wild-type gyrase strains. These results confirm that gyrase is the primary target of novobiocin and that the level of novobiocin needed to inhibit topo IV in vivo is close to the level which overcomes the resistance conferred by the alteration in gyrase.

FIG. 4.
Viability of gyrase and topo IV novobiocin-resistant strains in the presence of novobiocin. Cultures of the strains were diluted and plated onto increasing concentrations of novobiocin. The symbols correspond to the following acr strain genotypes: [filled triangle], ...

DISCUSSION

We created a mutation in the parE gene homologous to a commonly mutated residue in gyrB alleles conferring novobiocin resistance. In vitro, the ParE R132C protein exhibits a 40-fold increase in resistance to novobiocin. The ParE R132C alteration allows topo IV to retain its ability to relax positive supercoils in vivo at more than 20 times the concentration of novobiocin at which wild-type topo IV is inhibited. The mutation conferred no increase in viability in the presence of drug in an otherwise wild-type background and only a small increase in a gyrB234 background because levels of novobiocin that inhibit topo IV overcame the gyrase resistance mutation.

We conclude the following. First, topo IV is indeed a secondary target for novobiocin for E. coli. It will be interesting to determine whether the priority of targets is reversed for other bacteria, as is the case with quinolones (12, 34). Second, the mechanism of inhibition of topo IV by novobiocin is very likely the same as for gyrase, namely the blocking of ATP binding. The chief reason for concluding this is that alterations in the proteins conferring drug resistance are homologous and lie in equivalent positions near the ATP binding pockets of both enzymes. Third, E. coli does not readily mutate to high-level coumarin resistance by successive mutation of gyrase and topo IV, as has been observed with quinolones (18, 27, 43).

Our results highlight differences in how quinolone and coumarin drugs target topoisomerases. Because the novobiocin and ATP binding sites overlap, alteration of the protein to resistance reduces enzymatic activity. Thus, both gyrase and topo IV resistance come with the price of impaired growth. We observed this effect in two ways. First, the strain resistant for both topoisomerases produced only about 20% as many viable cells per optical density unit as the wild-type strain. Second, mutation of parE in a wild-type gyrase background actually increased the sensitivity of the otherwise wild-type strain to novobiocin (Fig. (Fig.44).

Another difference between coumarin and quinolone antimicrobial agents is in their relative affinities for gyrase and topo IV. The difference between the inhibition constants of gyrase and topo IV is much greater for coumarins than for quinolones (35). Additive increases in drug resistance by successive mutations require that the resistance conferred to the primary target overlap the sensitivity range of the secondary target. Thus, the similar inhibition constants of quinolones for gyrase and topo IV make it easier for these drugs to elicit a sequential resistance that may be unattainable for coumarins. We tried repeatedly, but unsuccessfully, to isolate a high-level novobiocin-resistant parE mutant strain by plating the gyrB234 strain on increased levels of novobiocin. The reasons for this are now clear. First, topo IV is much less novobiocin sensitive than gyrase, and the amount of novobiocin that inhibits topo IV overcame the resistance of gyrase. Second, the double mutant is compromised in growth.

While there are many sites in GyrA at which substitutions can confer quinolone resistance, only two sites have been identified in GyrB that confer coumarin resistance. This limitation on the number of available sites for effective alteration, and the deleterious nature of such changes, may make the development of high-level resistance to coumarin drugs through mutations in topoisomerase genes unfavorable. The use of coumarin drugs for clinical purposes has suffered due to the drugs' poor permeation in gram-negative bacteria and their toxicity in eukaryotes (32). However, the emergence of multidrug resistance in pathogenic bacteria has heightened interest in expanding the range of effective antimicrobial agents (3, 5). New drugs belonging to the coumarin family may yet be developed as important pharmaceuticals.

Novobiocin and other coumarin antibiotics have proved to be invaluable for dissecting the roles of topoisomerases in DNA replication (2, 24, 38). They have certain experimental advantages over quinolones that arise from the fate of quinolones to form difficult-to-repair lesions of DNA that poison the cell (13, 26, 42). Quinolone drugs cause double-strand breaks in DNA and induce the SOS response (8). Thus, novobiocin is a more desirable compound for selectively inhibiting topoisomerase activity without directly inducing DNA lesions (11).

A thorough understanding of the in vivo targets of coumarins is vital to the use of these drugs for research purposes. Here we have shown that the ParE protein can be altered to novobiocin resistance and that topo IV is a target of coumarin drugs in vivo. Inhibition of topo IV leads to the suppression of active positive supercoil relaxation, supporting a role for topo IV in the elongation step of DNA replication (24). We have created a useful cassette that can be used in any E. coli strain to ensure that gyrase, and not topo IV, is being inhibited by novobiocin.

Acknowledgments

This work was supported by National Institutes of Health grant GM31657. C.D.H. is a Howard Hughes Medical Institute predoctoral Fellow.

We are very grateful to D. Wigley for sharing with us his unpublished structure of ParE. We thank H. Hiasa for providing the ParE overexpression plasmid and for helpful discussion. We thank S. Kustu and H. Nikaido for strains.

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