J. Mol. Biol.

(1996) 258, 627637

DNA Gyrase and Topoisomerase IV on the Bacterial

Chromosome: Quinolone-induced DNA Cleavage
Chang-Rung Chen1, Muhammad Malik1, Michael Snyder2 and
Karl Drlica1*
1

Public Health Research

Institute, 455 First Avenue
New York, NY 10016, USA
2

Departments of Biology and

of Molecular Biophysics and
Biochemistry, Yale University
New Haven, CT 06520-8103
USA

DNA gyrase, the bacterial enzyme that supercoils DNA, is trapped on

chromosomal DNA by the 4-quinolone compounds, as druggyrase
complexes that contain DNA breaks. Examination of chromosomal DNA
extracted from Escherichia coli indicated that bacteriostatic concentrations of
oxolinic acid trap gyrase and block DNA synthesis without releasing
broken DNA from gyraseDNA complexes. Release, detected as free
rotation of DNA in the presence of an intercalating dye, occurred only at
high, bactericidal oxolinic acid concentrations. Release of DNA breaks and
cell death were both blocked by chloramphenicol, an inhibitor of protein
synthesis, suggesting that synthesis of additional protein activity is
required to free the DNA ends. Ciprofloxacin, a more potent quinolone,
released DNA breaks and killed cells even in the presence of
chloramphenicol. It is proposed that this second, chloramphenicol-insensitive mode for release of DNA breaks and cell killing arises from
dissociation of gyrase subunits. Ciprofloxacin also killed a gyrase (gyrA)
mutant resistant to the prototype of quinolone, nalidixic acid, and created
complexes on DNA detected by DNA fragmentation. This lethal effect of
ciprofloxacin was eliminated by additional mutations mapping in parC, one
of the two genes encoding topoisomerase IV. Thus, the fluoroquinolone
compounds have two intracellular targets. In the absence of the gyrA
mutation, the parC (CipR) allele did not by itself confer resistance to
ciprofloxacin, indicating that gyrase is the major quinolone target in E. coli.
These findings provide a molecular explanation for quinolone action in
bacteria and a new way to study topoisomerase IVchromosome
interactions.
7 1996 Academic Press Limited

*Corresponding author

Keywords: gyrase; topoisomerase IV; Escherichia coli; ciprofloxacin; lexA

gene

Introduction
Bacterial DNA topology is controlled by three
enzymes: DNA gyrase introduces negative supercoiling (Gellert et al., 1976a,b; Drlica & Snyder,
1978); DNA topoisomerase I counters the action of
gyrase to prevent the accumulation of excess
supercoiling (DiNardo et al., 1982; Pruss et al., 1982);
and DNA topoisomerase IV plays a central role in
the resolution of interlinked, replicated daughter
chromosomes (Kato et al., 1990; Adams et al., 1992).
Gyrase and topoisomerase IV are related, having
amino acid sequence similarity and similar doublePresent address: C.-R. Chen, Department of Biology,
New York University, Washington Square, New York,
NY 10003, USA.
00222836/96/19062711 $18.00/0

strand passage modes of action. In addition, both

are sensitive to the 4-quinolone class of antibacterial
compound in vitro (Gellert et al., 1977; Sugino et al.,
1977; Peng & Marians, 1993b; Hoshino et al., 1994).
We have been particularly interested in quinolone
sensitivity because the drugtopoisomerase interaction appears to be marked by DNA breakage that
can be monitored for study of topoisomerase
chromosome relationships (Drlica et al., 1990).
It has been known for many years that gyrase is
a physiological target of quinolone compounds in
Escherichia coli. Resistance mutations map in the
gyrase genes (Hane & Wood, 1969; Gellert et al.,
1977; Sugino et al., 1977), and the quinolone
compounds trap gyrase on DNA (Gellert et al., 1977;
Sugino et al., 1977) in parallel with rapid inhibition
of DNA synthesis (Snyder & Drlica, 1979). Indeed,
7 1996 Academic Press Limited

628
for several quinolone compounds, inhibition of
DNA synthesis occurs at the minimum inhibitory
concentration for growth (Chow et al., 1988), which
has been thought to equal minimum bactericidal
concentration. Inhibitors of RNA or protein synthesis prevent the prototype quinolone, nalidixic
acid, from quickly killing cells (Deitz et al., 1966);
consequently, rapid killing appears to arise from an
active process subsequent to the passive block of
DNA replication. Breakage of DNA would seem to
be involved, since quinolonegyraseDNA complexes contain broken DNA (Gellert et al., 1977;
Sugino et al., 1977; Snyder & Drlica, 1979). However,
a lethal role for DNA breaks has been unclear
because they have been observed only after
extraction of DNA under conditions of protein
denaturation. Nucleoid DNA remains supercoiled
when isolated from cells treated with concentrations
of oxolinic acid that fully inhibit DNA synthesis
(Snyder & Drlica, 1979) and apparently kill cells
(Chow et al., 1988). Thus, it has not been obvious
how quinolone compounds cause cell death. Below
we show that the release of DNA breaks from
gyraseDNA complexes, not inhibition of DNA
synthesis, correlates with killing of E. coli by oxolinic
acid.
A related issue concerns the ability of inhibitors
of RNA and protein synthesis to interfere with
killing by quinolone compounds. Rifampicin and
chloramphenicol are very effective at inhibiting the
lethal effect of nalidixic acid arising from nalidixic
acidgyraseDNA interactions. However, the lethal
effect of the potent fluoroquinolone ciprofloxacin is
only partially blocked by either rifampicin or the
presence of a nalidixic acid resistance (NalR ) allele
of gyrA, one of the two genes encoding gyrase
(Lewin et al., 1991). Since rifampicin completely
blocks the lethal effect of ciprofloxacin on a gyrA
(NalR ) mutant, protein synthesis-independent
killing involves gyrase. To account for the inability
of rifampicin to fully block the killing effect of
ciprofloxacin on wild-type cells, we postulate that
dissociation of gyrase subunits in ciprofloxacin
gyraseDNA complexes releases DNA ends, an
event expected to be lethal. Since the gyrA (NalR )
allele confers only partial protection from ciprofloxacin, this quinolone also has a non-gyrase target.
Below we present evidence that topoisomerase IV
is the non-gyrase target of ciprofloxacin. This
accounts for the susceptibility of gyrA (NalR )
strains to ciprofloxacin and provides a new way to
study topoisomerase IVchromosome interactions.

TopoisomeraseChromosome Interactions

Figure 1. Effect of chloramphenicol on viability and

DNA synthesis in the presence of oxolinic acid. A, Viable
cells. Aliquots of E. coli strain NI747, growing exponentially, were treated with the indicated concentrations of
oxolinic acid with (w) or without (W) treatment with
20 mg/ml chloramphenicol. Cells were then incubated for
2.5 hours at 37C, diluted into cold LB lacking antibiotics,
and plated. Viable colonies were determined after
overnight incubation at 37C. B, DNA synthesis. Aliquots
of E. coli strain NI747 were treated with the indicated
concentrations of oxolinic acid with (w) or without (W)
treatment with 20 mg/ml chloramphenicol. At various
times after addition of oxolinic acid, samples were
removed for a three minute labelling period with
3
H-labelled thymidine. Incorporation of radioactivity into
an acid-insoluble form drops within a few minutes after
quinolone treatment and reaches a plateau (Snyder &
Drlica, 1979). DNA synthesis rate at the plateau is
expressed as a percentage of the untreated control.

Results
Oxolinic acidgyraseDNA complex formation
is insufficient to kill cells
To connect chromosomal changes and lethal
effects caused by oxolinic acid, we first examined
the action of chloramphenicol, an inhibitor of
protein synthesis. When added to liquid cultures of
E. coli shortly before oxolinic acid, chloramphenicol

almost eliminated cell death (Figure 1A). However,

chloramphenicol had little effect on inhibition of
DNA synthesis by oxolinic acid (Figure 1B). In
addition, inhibition of DNA synthesis was more
sensitive to oxolinic acid than was survival. A drug
concentration sufficient to inhibit DNA synthesis by
90% in ten minutes had little effect on survival
during a 2.5 hour treatment (Figure 1A and B).

TopoisomeraseChromosome Interactions

629
Quinolone-induced release of DNA supercoils

Figure 2. Sedimentation of quinolone-generated DNA

fragments. Exponentially growing E. coli cells (strain
NI747) were treated with 3H-labelled thymidine for 30
minutes, and then oxolinic acid was added for ten
minutes. Cells were lysed, treated with 0.5% (w/v)
sodium dodecyl sulfate, and centrifuged into sodium
dodecyl sulfate-containing sucrose density-gradients
(Snyder & Drlica, 1979). Radioactivity in each fraction has
been normalized to the maximum value observed for
each gradient. A, 1 mg/ml oxolinic acid; B, 30 mg/ml
oxolinic acid. DNA from untreated cells sedimented about
2.5 times faster than that from cells treated with oxolinic
acid. Arrows indicate the position of 14C-labelled
bacteriophage T4 DNA included in each gradient.
Sedimentation was from right to left.

These data demonstrate that inhibition of DNA

synthesis is not by itself responsible for the
bactericidal effect of oxolinic acid.
Since inhibition of DNA synthesis parallels
formation of quinolonegyrase complexes with
chromosomal DNA (Snyder & Drlica, 1979), we
determined whether the same number of complexes
form at the low drug concentrations sufficient to
inhibit DNA synthesis as form at the high
concentrations needed for killing. No difference was
observed in the size of DNA fragments generated
by treatment of cells with oxolinic acid at 1 and
30 mg/ml when followed by protein denaturation
to release DNA ends constrained in druggyrase
DNA complexes (Figure 2). Thus, quinolone
gyraseDNA complex formation, as detected by
sodium dodecyl sulfate-dependent DNA fragmentation, is not sufficient to kill cells.

Treatment of cells with bacteriostatic concentrations of oxolinic acid (1 mg/ml) has little or no
effect on the sedimentation properties of isolated
nucleoids (Snyder & Drlica, 1979). To examine the
effect of high, bactericidal concentrations of oxolinic
acid, E. coli DNA was radioactively labelled by
growth in [3H]thymidine, cells were treated with 0,
5, 10, or 20 mg/ml oxolinic acid for ten minutes prior
to gentle lysis, and nucleoids were sedimented into
neutral sucrose density-gradients lacking detergent.
In each case discrete, rapidly sedimenting nucleoids
were observed, and little radioactivity was recovered as small DNA fragments at the tops of sucrose
density-gradients (Figure 3). Thus, the nucleoids
maintained much of their integrity even at high
quinolone concentration. However, oxolinic acid at
bactericidal concentrations (5 to 10 mg/ml) reduced
the nucleoid sedimentation rate from 1750 S to
about 1300 S. With twice as much drug, a shoulder
of more slowly sedimenting material could be seen
(Figure 3D). At very high concentrations of oxolinic
acid (50 to 75 mg/ml), the shoulder, which
sedimented at about 800 S, became more prominent
(Figure 4). Thus, treatment of cells with increasing,
bactericidal concentrations of oxolinic acid caused
two changes in nucleoid structure, conversion to a
1300 S form at intermediate drug concentrations and
to an 800 S form at high drug concentrations.
The decrease in sedimentation rate from 1750 S to
1300 S (Figure 3) was similar to that seen when
supercoils are relaxed by ethidium bromide or by
DNA nicks (Worcel & Burgi, 1972). To determine
whether supercoils were eliminated by bactericidal
concentrations of oxolinic acid, nucleoids were
sedimented into a series of sucrose gradients containing various concentrations of ethidium bromide.
Increasing concentrations of ethidium titrate negative supercoils and reduce the sedimentation
coefficient. At a critical ethidium concentration, all
supercoils are removed; further increases in dye
concentration introduce positive supercoils and
increase sedimentation rate. Although bacteriostatic
concentrations of oxolinic acid sufficient to inhibit
DNA synthesis by 95% (1 mg/ml) relaxed supercoiling slightly, as shown by a shift in the sedimentation
minimum to lower ethidium concentrations (Figure 5A), high concentrations of ethidium clearly
introduced positive supercoils, as shown by the
increase in sedimentation rate (Figure 5A, open
circles). These data indicate that DNA breaks,
presumably generated during quinolonegyrase
DNA complex formation and observed upon
treatment with sodium dodecyl sulfate (Figure 2),
were constrained and unable to release superhelical
tension. In contrast, bactericidal quinolone concentrations rendered nucleoid sedimentation independent of ethidium concentration (Figure 5B to D).
These are the results expected if DNA breaks
created by oxolinic acid at high concentration were
released and allowed free rotation of the DNA
strands, since treatment with DNase creates a

630

TopoisomeraseChromosome Interactions

similar response (Worcel & Burgi, 1972). It is

unlikely that the flattening of the ethidium titration
curves seen in Figure 5 arose from heterogeneity
within or among nucleoids, since plasmid DNA
known to be topologically heterogeneous shows a
distinct increase in sedimentation rate at 4 mg/ml
ethidium bromide (Drlica et al., 1988). The experiment shown in Figure 5D showed no nucleoid
sedimentation increase even at 6 mg/ml ethidium
bromide (data not shown).
Since chloramphenicol blocked the lethal effect of
oxolinic acid (Figure 1A), we determined whether
parallel changes in chromosome structure occurred
in the presence of chloramphenicol. Nucleoids from
cells treated with both chloramphenicol and oxolinic
acid displayed a distinct sedimentation minimum
(Figure 6A, open circles), indicating that chloramphenicol treatment blocked the release of supercoil
constraint associated with oxolinic acid treatment.
Since the same result would be seen if inhibition of
protein synthesis eliminated gyrasechromosome
interactions, cells were treated with chloramphenicol for 30 minutes, oxolinic acid was added for
another ten minutes and cells were lysed in the
presence of sodium dodecyl sulfate to release DNA
breaks from gyrase-mediated constraint. When the
DNA fragments were sedimented into sucrose
density-gradients, they exhibited only a modest
(14%) increase in average sedimentation rate relative
to fragments from cells treated with oxolinic acid
alone (data not shown). Thus, oxolinic acid-induced
complexes are formed in the presence of chloramphenicol (they formed in undiminished numbers
after inhibition of protein synthesis by 75 minutes
of starvation for essential amino acids (data not
shown)). Taken together, these data support the idea
that intracellular release of DNA ends, not simply
formation of quinolonegyraseDNA complexes,
accounts for the lethal effect of oxolinic acid.
The more recently discovered fluoroquinolone
compounds, such as ciprofloxacin, possess an
additional mode of killing that does not require ongoing protein synthesis (Howard et al., 1993a,b).
Since gyrase can participate in quinolone-stimulated
subunit exchange (Ikeda, 1994), it seemed possible
that the second mode of ciprofloxacin action
might involve direct dissociation of quinolone
gyraseDNA complexes. As a test for this idea,
E. coli was treated with 1 mg/ml ciprofloxacin and
supercoil constraint was examined in nucleoids.
Ethidium bromide titration curves of nucleoids
were flat, whether or not cells were treated with
chloramphenicol (Figure 6B). At this concentration
of ciprofloxacin, chloramphenicol protected fewer
than 1% of the cells from killing by ciprofloxacin
(Figure 7). Thus, cell death parallels the freedom of
DNA strands to rotate, which is presumably due to
Figure 3. Effect of oxolinic acid on nucleoid sedimentation. 3H-labelled nucleoids were obtained from strain
NI747 treated with oxolinic acid at 0 (A), 5 mg/ml (B),
10 mg/ml (C), or 20 mg/ml (D) for ten minutes by cell lysis
in the absence of ionic detergents (Drlica & Snyder, 1978).
Nucleoids were then sedimented into 10% to 30% sucrose

density-gradients at 7000 rpm, and gradients were fractionated from the bottom of the centrifuge tubes. The
broken line indicates the position of 14C-labelled bacteriophage T4B added to each gradient as a sedimentation
marker. Sedimentation was from right to left.

TopoisomeraseChromosome Interactions

Figure 4. Slow-sedimenting species of nucleoid

generated by treatment of cells with oxolinic acid at high
concentration. 3H-labelled nucleoids were obtained from
strain NI747 treated for ten minutes with oxolinic acid at
0 (A), 50 mg/ml (B), or 75 mg/ml (C) as described for
Figure 3. Nucleoids were then sedimented into 10% to
30% sucrose density-gradients at 7000 rpm. The position
of 14C-labelled bacteriophage T4B is indicated by arrows.
Sedimentation is from right to left.

the release of DNA breaks from constraint by

gyrase. Figure 7 also revealed that ciprofloxacin at
low concentration behaves much like the less potent
oxolinic acid at high concentration, since chloramphenicol was very effective at blocking the bactericidal action at low concentrations of ciprofloxacin.
Thus, increasing the concentration or potency of a
quinolone appears to shift its mode of action from
one requiring protein synthesis to one that does not.
DNA topoisomerase IV is an intracellular
target of ciprofloxacin
Since quinoloneresistant mutants of Staphylococcus aureus contain mutations in a gene encoding

631

Figure 5. Effect of oxolinic acid on supercoiling in

bacterial nucleoids. 3H-labelled nucleoids were obtained
from cells treated with oxolinic acid for ten minutes at 0
(W) or 1 mg/ml (w) (A), 5 mg/ml (B), 10 mg/ml (C), or
20 mg/ml (D). Sedimentation coefficients, relative to
14
C-labelled bacteriophage T4B, were estimated from
centrifugation into sucrose density gradients similar to
those shown in Figure 3 but containing the indicated
concentrations of ethidium bromide (EBr; Hsieh et al.,
1991).

topoisomerase IV (Ferrero et al., 1994), we considered the possibility that topoisomerase IV might
be a second target of the quinolone compounds. To
test this idea, we prepared E. coli mutants and
analyzed them by transduction of parC, one of the
genes encoding topoisomerase IV. Two strains, CR1
and CR3, were isolated by plating the gyrA-307
(NalR ) strain GP201 on agar containing 0.5 mg/ml
ciprofloxacin, a concentration that prevented growth

632

TopoisomeraseChromosome Interactions

Figure 7. Effect of chloramphenicol on bacterial

survival during treatment with ciprofloxacin. E. coli strain
NI747, growing exponentially, was treated with (W) or
without (w) chloramphenicol (20 mg/ml) for ten minutes
followed by an additional ten minutes incubation with
the indicated concentrations of ciprofloxacin. Cells were
diluted and grown on agar plates to determine the
number of viable cells.

Effect of ciprofloxacin on gyrA and

parC mutants

Figure 6. Effect of chloramphenicol and oxolinic acid or

ciprofloxacin on DNA supercoiling in bacterial nucleoids.
A, Nucleoids were obtained from E. coli cells (strain
NI747) and sedimentation analysis was performed as
described for Figure 5, except that cells were treated with
20 mg/ml chloramphenicol for 30 minutes followed by
oxolinic acid at 8 mg/ml for ten additional minutes (w).
As controls, nucleoids were obtained from untreated cells
(w) or from cells treated only with 8 mg/ml oxolinic acid
(W). B, Nucleoids from E. coli strain NI747 were obtained
and subjected to sedimentation analysis as described for
Figure 5, except that cells received 20 mg/ml chloramphenicol for 30 minutes prior to 1 mg/ml ciprofloxacin for
ten minutes (w) or only 1 mg/ml ciprofloxacin for ten
minutes (W).

of strain GP201. To map the mutations conferring

ciprofloxacin resistance, transductional analysis
was performed using a bacteriophage P1 lysate
prepared in strain KD1100 (parC-1215 (Ts)
metC::Tn10 (TetR )). After selecting for TetR, cotransduction frequencies with TetR were between 50%
and 60% for temperature and ciprofloxacin sensitivity. Ts and CipS were 96% and 86% cotransducible
with each other for recipient strains CR1 and CR3,
respectively. Thus, by this criterion ciprofloxacin
resistance maps at or very close to parC.

Chloramphenicol provided partial protection

from ciprofloxacin at 0.5 mg/ml (Figure 8A, strain
DM4100), as did a gyrA (NalR ) mutation (Figure 8A,
strain GP201). Full resistance was conferred to the
gyrA (NalR ) mutant by treatment with chloramphenicol (Figure 8A). These data indicate that
gyrase is a target of the chloramphenicol-insensitive
mode of bacterial killing by ciprofloxacin described
above. These data also support the assertion that
cells contain a non-gyrase activity whose susceptibility to ciprofloxacin is blocked by inhibition
of protein synthesis. Addition of a parC (CipR )
mutation to the gyrA (NalR ) mutant eliminated
susceptibility to the bactericidal effects of
ciprofloxacin (Figure 8B, strain CR1), consistent
with topoisomerase IV acting as a target of
ciprofloxacin. Taken together, these data make it
likely that chloramphenicol blocks lethal effects
associated with topoisomerase IVciprofloxacin
interactions. Unlike the situation with wild-type
cells (strain DM4100), the lethal effects associated
with topoisomerase IV accumulated slowly, since
there was a distinct lag before killing commenced
with the gyrA (NalR ) strain GP201 (Figure 8A).
Introduction of the lexA-3 allele, which prevents
induction of the SOS response, eliminated the lag
(compare strains GP201 and KD1237, Figure 8C).
Thus, one or more components of the SOS response
retard the lethal effects of ciprofloxacintopoisomerase IV interactions.

633

TopoisomeraseChromosome Interactions

Figure 8. Protection from the bactericidal action of ciprofloxacin by chloramphenicol and topoisomerase mutations.
Exponentially growing E. coli cells were treated with 0.5 mg/ml ciprofloxacin. Chloramphenicol treatment, when
administered, began five minutes prior to ciprofloxacin. At the indicated times, cells were diluted and grown on agar
plates to determine the number of viable cells. A, Effect of a gyrA (NalR ) mutation. E. coli strains were DM4100 (wild
type (w); DM4100 treated with 20 mg/ml chloramphenicol (W); GP201 (gyrA-307 (q); GP201 (gyrA-307 treated with
20 mg/ml chloramphenicol (Q)). B, Effect of gyrA (NalR ) and parC (CipR ) mutations. E. coli strains were GP201 (gyrA-307
(q)) and CR1 (gyrA-307 parC-2101 (r)). C, Effect of lexA-3 (Ind ) and gyrA (NalR ) mutations. E. coli strains were GP201
(gyrA-307 (q)) and KD1237 (gyrA-307 lexA-3 (R)). D, Effect of a parC (CipR) mutation. E. coli strains were DM4100
(wild type (w)); DM4100 treated with 20 mg/ml chloramphenicol (W); and CR104 (parC CipR (r)). E, Effect of
chloramphenicol and a parC (CipR ) mutation. E. coli strains were DM4100 (w); DM4100 treated with 20 mg/ml
chloramphenicol (W); (GP201 (gyrA-307 (q)); and CR104 (parC CipR) treated with 20 mg/ml chloramphenicol (R).

By itself, the parC (CipR ) allele showed little or no

protective effect against ciprofloxacin (Figure 8D,
strain CR104), consistent with the hypothesis that
gyrase is the primary target of the antibiotic. Since
chloramphenicol provided more protection than the
parC mutation (Figure 8D, compare strain CR104
with strain DM4100 plus chloramphenicol), inhibition of protein synthesis does not confer protection only from ciprofloxacintopoisomerase IV
DNA complexes. It is likely that most of the
protective effect of chloramphenicol arises from
interference with the release of DNA breaks from
ciprofloxacingyraseDNA complexes, as is the case
for oxolinic acidgyraseDNA complexes.
Shortly after administration of ciprofloxacin,
strain CR104 (parC CipR ), when also treated with
chloramphenicol, was less susceptible to ciprofloxacin than the wild-type strain DM4100 treated
with chloramphenicol (Figure 8E). After about 60
minutes, however, strain CR104 was killed. A
similar response, but with a longer lag, was seen
with the gyrA (NalR ) mutant (Figure 8E). If gyrase
and topoisomerase IV are the only targets of ciprofloxacin, as suggested by Figure 8B, then the
response seen with strain CR104 (parC CipR ) treated
with chloramphenicol reflects the putative gyrase
dissociation mode of killing. The response observed
with the gyrA (NalR ) mutant GP201 would represent
the topoisomerase IV-mediated effect. It appears
that potentially lethal events due to ciprofloxacin
accumulate more quickly with gyrase than with

topoisomerase IV, consistent with gyrase being the

major target for this drug.
Sedimentation analysis revealed that DNA fragments generated by treatment of wild-type cells
with 1 mg/ml ciprofloxacin were smaller than those
observed with oxolinic acid (Figure 9), consistent
with ciprofloxacin trapping both gyrase and
topoisomerase IV on the chromosome. As expected,
the size of the DNA fragments created by
ciprofloxacin with the parC (CipR ) gyrA+ mutant
CR104 was the same as that following treatment of
wild-type cells with oxolinic acid (data not shown).
For the gyrA (NalR ) strain GP201, preliminary
sedimentation analysis showed that the number
average molecular weight of ciprofloxacin-induced
fragments was twice that seen following oxolinic
acid treatment of the wild-type strain DM4100 (data
not shown). Thus, topoisomerase IV appears to be
trapped at more widely dispersed sites on the
chromosome than is gyrase.

Discussion
Once it became clear that higher oxolinic acid
concentrations were required to kill cells than to
block DNA synthesis, connections emerged between lethal action and DNA lesions due to this
drug. Our current view is sketched in Figure 10.
Moderate concentrations of oxolinic acid (1 mg/ml)
trap gyrase on DNA such that the ends of broken
DNA are constrained by gyrase (Figure 10, step b).

634

Figure 9. Sedimentation of ciprofloxacin-generated

DNA fragments. Exponentially growing E. coli strain
NI747 was treated with 3H-labelled thymidine for 30
minutes and then 0.5 mg/ml ciprofloxacin (W) or
30 mg/ml oxolinic acid (w) for 10 minutes. Cells were
lysed, treated with 0.5% (w/v) sodium dodecyl sulfate,
and centrifuged into separate sodium dodecyl sulfatecontaining sucrose density-gradients (Snyder & Drlica,
1979). Each density-gradient contained 14C-labelled
bacteriophage T4B DNA as a sedimentation marker that
allowed the gradients to be superimposed in the Figure.
DNA radioactivity in each fraction has been normalized
to the maximum value observed for each gradient. Arrow
indicates the position of bacteriophage T4 DNA.
Sedimentation was from right to left.

Supercoils remain, and only after treatment with

sodium dodecyl sulfate is fragmentation revealed
(Figures 2A and 5A; Snyder & Drlica, 1979). Since
removal of quinolone reverses inhibition of DNA
synthesis (Deitz et al., 1966) and DNA fragmentation
(data not shown), moderate (1 mg/ml) quinolone
concentrations are only bacteriostatic. Raising the
oxolinic acid concentration to 5 to 20 mg/ml had no
effect on the number of druggyraseDNA complexes on chromosomal DNA (Figure 2). It did,
however, lead to a decrease in nucleoid sedimentation rate (Figure 3), to the release of DNA breaks,
as seen by loss of supercoil constraint (Figure 5) and
to cell death. Additional support for the presence of
DNA ends in nucleoids obtained from cells treated
with oxolinic acid emerged from the sensitivity of
nucleoid sedimentation rate to rotor speed: nucleoids sedimented at 800 S rather than 1300 S when
the rotor speed was increased from 7000 rpm to
17,000 rpm (data not shown). This type of rotor
speed effect is characteristic of long, extended DNA
containing ends (Zimm, 1974; Zimm & Schumaker,
1976).
The bactericidal action of nalidixic and oxolinic
acids occurs primarily through a pathway that is
inhibited by chloramphenicol (Figure 10, step c).
Chloramphenicol prevented the oxolinic acid-induced loss of supercoil constraint (Figure 6A) and
cell death (Figure 1A), but not inhibition of DNA
synthesis (Figure 1B; Deitz et al., 1966). We postulate
that a repair factor is involved in this pathway. The

TopoisomeraseChromosome Interactions

Figure 10. Schematic representation of events involved

in quinolone-mediated cell death. Gyrase and DNA
interact (a) in such a way that 4-quinolone compounds can
trap (b) a reaction intermediate that blocks DNA synthesis
and cell growth. DNA in the reaction intermediate is
broken, but the ends are restrained by gyrase. The DNA
ends can be released by two pathways, both of which lead
to cell death. Pathway c is inhibited by chloramphenicol.
It is postulated that this pathway is mediated by a repair
factor that removes quinolonegyrase complexes from
DNA. Such a factor has not been identified. At high
concentrations of potent quinolone compounds the DNA
ends can be released by a chloramphenicol-insensitive
pathway (d). It is postulated that this pathway involves
gyrase subunit dissociation. The presence of gyrase on
DNA ends has not been demonstrated. A similar series of
complexes and broken DNA products may form when
quinolone compounds interact with topoisomerase IV.

putative factor is independent of the SOS response,

since the lethal effects of nalidixic acid are
unaffected by the lexA-3 mutation (Lewin et al.,
1989), which prevents the SOS response (Rinken &
Wackernagel, 1992). Since chloramphenicol rapidly
blocks the lethal effect of nalidixic acid even when
added two to three hours after the quinolone (Deitz
et al., 1966), it is likely that the putative factor turns
over rapidly.
Fluoroquinolone compounds; such as ciprofloxacin, also have gyrase as a target and behave in
a way similar to nalidixic and oxolinic acids;
however, ciprofloxacin causes two additional events
to occur. One is a chloramphenicol-insensitive mode
of killing, which we postulate to involve dissociation of the gyrase subunits that constrain DNA
ends in ciprofloxacingyraseDNA complexes (Figure 10, step d). The idea of subunit dissociation
emerged from Ikedas observation that certain
classes of illegitimate recombination can be explained by gyrase subunit exchange and that
quinolone compounds stimulate this exchange
(reviewed by Ikeda, 1994). Support for the gyrase
dissociation idea is provided by the inability of
chloramphenicol to block either the lethal action of
ciprofloxacin (Figure 7) or the ciprofloxacin-depen-

635

TopoisomeraseChromosome Interactions

dent flattening of nucleoid ethidium titration curves

(Figure 6B). Evidence for gyrase involvement comes
from the ability of a gyrA (NalR ) mutation to
eliminate the chloramphenicol or rifampicin-insensitive mode of killing by ciprofloxacin (Figure 8A;
Lewin et al., 1991). Repair or prevention of DNA
damage due to subunit dissociation appears to
involve the SOS response, since a lexA-3 mutant is
hypersensitive to ciprofloxacin (Howard et al., 1993)
but not to nalidixic acid (Lewin et al., 1989).
Oxolinic acid may also participate in the putative
subunit dissociation mode, since this compound
stimulates a gyrase-dependent form of illegitimate
recombination and deletion formation that is most
easily interpreted in terms of gyrase subunit
dissociation and exchange (Ikeda et al., 1981, 1982;
Miura-Masuda & Ikeda, 1990). If this interpretation
is correct, a physical manifestation of subunit
dissociation may be the change in nucleoid
structure that decreases sedimentation rate from
1300 S to 800 S (Figure 4). This possibility is being
examined.
The second event seen with ciprofloxacin is
trapping of topoisomerase IV in complexes on DNA.
Transductional analysis, in which the recipient
strain was a gyrA (NalR ) mutant that had acquired
additional, spontaneous resistance to ciprofloxacin
(Figure 8B), showed that the additional ciprofloxacin resistance could be eliminated by replacement of parC, one of the two genes encoding
topoisomerase IV. A similar conclusion was reached
in concurrent work using strains constructed to
contain parC mutations (Khodursky et al., 1995). The
hypothesis of dual ciprofloxacin targets explains
why DNA cleavage fragments generated by
ciprofloxacin were smaller than those produced by
oxolinic acid (Figure 9). A chloramphenicol-inhibited mode of killing by ciprofloxacintopoisomerase
IV interactions would also explain why chloramphenicol protects gyrA (NalR ) mutants from the
lethal action of ciprofloxacin (Figure 8A).
Primary resistance to ciprofloxacin maps in gyrA
rather than in parC (gyrA NalR mutations typically
reduce susceptibility by 30 to 40-fold while a parC
CipR allele by itself provided no protection from
ciprofloxacin; Figure 8D). Why this occurs is not
obvious, since gyrase from E. coli is sometimes only
slightly more sensitive to fluoroquinolone compounds in vitro than is topoisomerase IV (Peng &
Marians, 1993b; Hoshino et al., 1994; Khodursky
et al., 1995). The two enzymes probably play very
different roles in chromosome topology. Gyrase is
associated with replication forks (Drlica et al., 1980)
and has access to most of the chromosome (Snyder
& Drlica, 1979; Franco & Drlica, 1988), presumably
to maintain appropriate levels of superhelical
tension. Topoisomerase IV is involved in decatentation (Adams et al., 1992), and it may be membranebound (Kato et al., 1992). Thus, the enzymes, when
bound to the chromosome, may differ in their
accessibility to the quinolone compounds and to the
recA-mediated recombinational processes important for survival following quinolone treatment

(Urios et al., 1991; Howard et al., 1993). Explanations

of the differences that focus on lethal, gyrase-mediated cleavage at replication forks (Khodursky et al.,
1995) are less likely because inhibition of DNA
synthesis is reversible (bacteriostatic) and fails to
correlate with lethal events. Cases in which
topoisomerase IV is the primary target, such as seen
with Staphylococcus aureus (Ferrero et al., 1994, 1995),
probably represent situations in which gyrase is
naturally resistant to quinolone compounds.
We currently favor the idea that the quinolone
compounds act on gyrase and topoisomerase IV in
a similar way and that the scheme shown in Figure 10 applies to both. According to this idea, the
bactericidal effect of ciprofloxacin with strain GP201
(gyrA NalR ) is due to an interaction with
topoisomerase IV. After an initial delay, cell death
occurs at the same rate as seen with wild-type cells.
Thus ciprofloxacintopoisomerase IV interactions
are bactericidal. Chloramphenicol eliminates the
bactericidal effect (Figure 8A), indicating that
much of the topoisomerase IV-mediated effect is
by pathway c (Figure 10). Since induced repair
functions contribute to bactericidal kinetics (Figure 8C), a complete explanation of the kinetic
response is likely to be complex.
According to the scheme in Figure 10, pathway d
is responsible for the lethal effect of ciprofloxacin
with strain CR104 (parC CipR ) when also treated
with chloramphenicol. In this case the target is
gyrase. Since inhibition of protein synthesis blocks
induced repair functions, the biphasic response
(Figure 8E) suggests that the putative gyrase
subunit dissociation mode kills cells after a delay.
Explaining this delay requires additional experimentation.
Preliminary examination of DNA fragments
obtained following ciprofloxacin treatment of a gyrA
(NalR ) mutant suggests that topoisomerase IV
interacts with the chromosome at widely dispersed
sites. This would allow topoisomerase IV to
decatenate DNA during replication (Peng &
Marians, 1993a). Through its association with
membrane (Kato et al., 1992), topoisomerase IV
might also serve to attach the chromosome to the
membrane (Drlica et al., 1978). Gyrase would then
specialize in maintaining superhelical tension
(Gellert et al., 1976a; Drlica & Snyder, 1978) and in
relaxing torsional tension arising from replication
fork movement (Drlica et al., 1980). Whether gyrase
normally supplements the decatenating activity of
topoisomerase IV is not known (Steck & Drlica,
1984; Bliska & Cozzarelli, 1987; Kato et al., 1990;
Adams et al., 1992).

Materials and Methods

Bacteriological methods and chemicals
The Escherichia coli K-12 strains studied were AQ8300
(lexA-3 (Ind ) malF3089::Tn10) obtained from T. Kogoma,
DM4100 (Sternglanz et al., 1981), NI747 (Gellert et al.,
1976a), GP201 (gyrA-307 NalR (Pruss et al., 1986)), and

636
NK6027 (metC::Tn10, obtained from B. Bachmann). CR1
and CR3 were spontaneous ciprofloxacin-resistant mutants of strain GP201; CR104 was a gyrA+ zeg-298::Tn10
transductant of CR1 isolated by screening for sensitivity
to nalidixic acid. For DNA cleavage studies, cells were
grown in M9 medium (Miller, 1972) supplemented with
40 mg/ml thymine, 1 mg/ml vitamin B1, and 60 mg/ml
leucine, isoleucine, valine, and threonine for strain NI747;
the only amino acid supplement for GP201 was cysteine.
For all other experiments growth was in LB medium
(Miller, 1972). Bacteriophage P1-mediated transduction
(Wall & Harriman, 1974) was used to construct a
metC::Tn10 parC-1215 (Kato et al., 1988) derivative of
DM4100 (KD1100), a lexA-3 derivative of GP201
(KD1237), and to map ciprofloxacin resistance. Cells were
treated with chloramphenicol (20 mg/ml) five minutes
before the addition of quinolone, unless otherwise
indicated, and this was continued for the duration of the
quinolone treatment. Radiolabelled compounds were
obtained from Amersham Chemical Co. Chloramphenicol, nalidixic acid, oxolinic acid, and ciprofloxacin were
products of Sigma Chemical Co.
Detection of quinolone-induced DNA cleavage
Release of chromosomal DNA breaks was monitored
by sedimenting 3H-labelled nucleoids in 10% to 30%
(w/v) sucrose density-gradients containing various
concentrations of ethidium bromide (Hsieh et al., 1991) in
the absence of sodium dodecyl sulfate. Breakage of
chromosomal DNA in the presence of sodium dodecyl
sulfate was monitored by sedimentation of DNA into 5%
to 20% (w/v) sucrose gradients containing sodium
dodecyl sulfate, following treatment of cell lysates with
sodium dodecyl sulfate (Snyder & Drlica, 1979).
Radioactive labelling, centrifugation conditions, gradient
fractionation, and radioactive counting were as described
(Drlica & Snyder, 1978; Snyder & Drlica, 1979). Centrifugation was performed with Beckman SW50.1 rotors.

Acknowledgements
We thank Drs R. Burger, M. Gennaro, and S. Kayman
for critical comments on the manuscript, J.-I. Kato and
T. Kogoma for providing strains carrying parC-1215
and lexA-3 alleles, respectively, and N. Cozzarelli for
communicating experimental results prior to publication.
A portion of the work was performed at the University of
Rochester. Support was from NIH grant AI35257.

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Edited by M. Gottesman
(Received 24 July 1995; received in revised form 5 February 1996; accepted 19 February 1996)