DNA supercoiling in
Escherichia coli
is under tight and subtle
homeostatic control, involving gene-expression and metabolic
regulation of both topoisomerase I and DNA gyrase
Jacky L. Snoep
1,2
, Coen C. van der Weijden
1
, Heidi W. Andersen
2,3
, Hans V. Westerhoff
1,4
and Peter Ruhdal Jensen
3
1
Departments of Molecular Cell Physiology and Mathematical Biochemistry, BioCentrum Amsterdam, Free University, Amsterdam,
the Netherlands;
2
Department of Biochemistry, University of Stellenbosch, South Africa;
3
Section of Molecular Microbiology,
Biocentrum, Technical University of Denmark, Lyngby, Denmark;
4
Stellenbosch Institute for Advanced Study, South Africa
DNA of prokaryotes is in a nonequilibrium structural
state, characterized as ÔactiveÕ DNA supercoiling. Altera-
tions in this state aect many life processes and a
homeostatic control of DNA supercoiling has been sug-
gested [Menzel, R. & Gellert, M. ( 1983) Ce ll 34, 105±113].
We here report on a new method for quantifying home-

constraint does not depend on the c ontinuous expenditure
of ATP. The r emaining supercoils are maintained actively at
the cost of ATP hydrolysis, via topoisomerase activities.
Four topoisomerases h ave been identi®ed in Escherichia coli
(reviewed in [2]). Topoisomerase I [3,4] and DNA gyrase
(topoisomerase II) are mostly held responsible for main-
taining the supercoiled state of the DNA while topoisom-
erase I II and IV manage the decatenation reactions.
A recent publication suggested that topoisomerase IV may
also be important for the relaxation of DNA supercoiling
[5].
The importance of DNA gyrase and topoisomerase I for
supercoiling has been shown in studies involving mutants
with ac tivities differing greatly from the wild-t ype ac tivity.
Such studies cannot be used to assess the homeostasis of
supercoiling in the physiological situation, where the
response to smaller challenges is important. When chal-
lenged suf®ciently, all systems will respond in drastic
manners, or fail. It may well be that a system is robust
with respect to small challenge s, whilst i t fails to deal with
the same but larger challenges, or vice versa.
DNA gyrase activity is known to be controlled home-
ostatically [6], but the extent o f this control a nd its
implications for the h omeostatic control of s upercoiling
itself, have not been quanti®ed. In general, homeostasis can
be conferred via changes in enzyme activity (e.g. due to
sensitivities for substrate, product or allosteric effectors) or
via c hanges in enzyme concentration transferred through
gene expression regulation. T he activities of both DNA
gyrase and topoisomerase I depend on the level of

Bacterial strains
The cloning work was performed in the strain DH5a or
JM105 [12,13]. Chromosome integration was performed in
strain MC1000 [14].
Growth of cultures
In th e t opoisomerase I and DNA gyrase modulation
experiments cells were pregrown in Mops (40 m
M
,
pH 7.4) minimal salts medium [15] containing 0.5% w/v
glucose, tricine ( 4 m
M
), valine, le ucine a nd isoleu cine
(40 lgámL
)1
each), thiamine (10 lgámL
)1
) and ampicillin
as antibiotic marker for pBR322 (100 lgámL
)1
)atthe
relevant isopropyl thio-b-
D
-galactosidase (IPTG) concen-
tration. After over night growth, the cells were diluted in the
same medium to an D
540
of 0.005 and growth was followed
for at least ®ve generations before sampling. All samples
were withdrawn between D

Topoisomerase concentration
The t opoisomerase I and DNA gyrase content of the cells
was estimated by quantitative W estern blotting using a n
antibody against topoisomerase I and GyrA subunit,
respectively. P uri®ed topoisomerase I and gyrase were
subjected to SDS/PAGE. After subsequent blotting to
nitrocellulose and Ponceau staining [Ponceau-S, 0.2% in
3% trichlororacetic acid (Serva)] the topoisomerase I and
gyrase A bands were cut out and ground. Polyclonal
antibodies were raised by Eurogentec by immunizing
rabbits with the ground fragments.
Construction of the plasmid used for the integration
at the
topA
locus pHA2
A 1549-bp PCR fragment c ontaining the DNA region
upstream of topA,thetopA promoter and the N-terminal
part of topA was ampli®ed using primers ECTOPA,
accession number X04475, bp322± 342, i.e. 5¢-CGAA
GAAGGGCGGGGAGAAAT-3¢ +bp1870±1850, i.e. 5¢-
TCCATAGCAGCGGCGAAACCA-3¢ and chromosomal
DNA from strain LM1237 [17] as a template. The PCR
fragment was subsequently digested with the enzymes DraI
and EcoRV and a 842-bp fragment containing the DNA
region upstream of topA and the topA promoter was
isolated and inserted into pUC19 (New England Biolabs)
digested with SmaI, resulting in the plasmid pHA2.
PHA5
The 1549-bp PCR f ragment described above w as digested
with EcoRV and SspI and a 572-bp fragment containing the

the blunted Kpn I±BamHI sites, resulting in t he plasmid
pTOPA2
TS
.
PTOPA2A5
TS
pTOPA2
TS
was digested ®rst with PstI and then with EcoRI
(partial digest), which removes the N-terminal part of the
Ó FEBS 2002 Control of DNA supercoiling by topoisomerase I (Eur. J. Biochem. 269) 1663
gyrA gene. Subsequently a 625-bp EcoRI±PstIfragment
from pHA5 containing the N -terminal part of topA was
inserted. T his resulted i n the plasmid pTOPA2A5
TS
,in
which the pA1lacO-1 promoter a nd the lacI
q1
gene are
surrounded by a DNA fragment originating from upstream
the topA gene and a fragment containing the N-terminal
part of the topA gene.
Replacement of the chromosomal
topA
promoter
with an inducible
lac
-type promoter and a
lacI
q1

as is shown in Fig. 1. I n the absence of IPTG the expression
was very low (2±5% of wild-type). Precise modulation of
expression around the wild-type level (at % 40 l
M
IPTG),
but also over-expression up to 20 times w ild-type was
possible. At any given IPTG concentration no signi®cant
dependence of topoisomerase concentration on cell density
was detected, in the range of cell concentrations represented
by D
540
 0.2±0.4, indicating a constant expression level of
the enzyme ( data not shown; cf [19]). Under these conditions
we should be able t o ask how readily DNA supe rcoiling is
perturbed by changes in topoisomerase I activity.
Is DNA supercoiling readily compromised
by topoisomerase I?
From t he p lot of aLk vs. the topoisomerase I c oncentration
(Fig. 2 ), it can be deduced that supercoiling is not very
sensitive for changes in topoisomerase I activity. Over a
thousand-fold range of expression of topoisomerase I the
aLk varied by no more than six linking numbers, i.e.
between )3 and +3 linking numbers relative to the )13
active links of the same plasmid in wild-type cells. Figure 2A
shows that at very low activities of topoisomerase I the
DNA supercoiling d epended even more weakly, if at all, on
the enzyme. At wild-type expression levels, the dependence
appeared to be stronger.
Fig. 1. IPTG induction of topoisomerase I expression. E. coli strain
HWA36 was inc ub ated with I PTG a t c oncentrations ranging fro m 0 to

b
1  e
Àtopoisomerase IÀc
d
with a  )173.219,
b  163.529, c  ) 5.2409, d  1.6430, long dash, aLk 
a  cÁlntopoisomerase I
1  bÁlntopoisomerase IdÁlntopoisomerase I
2
with a  )13.460, b  )0.0125,
c  1.3983, d  0.0072. (B) Shown a s an insert is the c ontrol of
topoisomerase I on DNA supercoiling. Inherent control c oecients
are calculated by multiplying the derivative of the ®tted c urves in
(A) at each point of the graph with the quotient of the respective
x/y coordinates. Thus the control coecient de®ned as
c
aLk
topoisomerase I

daLk
dtopoisomerase I
Á
topoisomerase I
aLk
is obtained. A t w ild-type
topoisomerase concentration an inherent control coecient of )0.14
was c alculated. `topoi somerase I ' refers to the concentration of
topoisomerase I relative to the wild-type.
1664 J. L. Snoep et al. (Eur. J. Biochem. 269) Ó FEBS 2002
How strong or weak the effect of topoisomerase I on

tested, topoisomerase I never had a high control on DNA
supercoiling. Also when the DNA became quite relaxed , its
control remained well below 0.2: DNA supercoiling is not
readily compromised by extra topoisomerase I.
Homeostasis of growth rate
Under t he conditions tested the s peci®c growth rate of
E. coli strain MC1000 was 0.93 h
)1
( 0.03) and was
observed to be almost in sensitive to a modulation of
topoisomerase I around its wild-type expression level. Only
at very low and very high expression levels was the growth
rate reduced by at most 25% (data not shown). The
dependence o f g rowth r ate on topoisomerase activity
around the physiological state was estimated as precisely
as possible: the c orresponding control c oef®cient w as as low
as 0.03, re¯ecting that a doubling of t he topoisomerase
activity decreased growth rate by a mere 3%.
Homeostasis through supercoiling dependent DNA
gyrase expression
The e xpression of the DNA gyra se g enes is alte red b y
mutations th at strongly affect DNA supercoiling [6,9]. If in
our experiments the concentration of DNA gyrase changed
in proportion to the change in concentration o f topoisom-
erase I, one should expect supercoiling t o be virtually
unaffected by the modulation o f topoisomerase expression
levels; i ndeed such a compensation mechanism could
explain the observed homeostasis. Accordingly we meas-
ured the cellular concentration of DNA gyrase at the
various expression levels of topoisomerase I.

elasticity coef®cient suggests that gyrase gene expression was
suf®ciently sensitive to DNA supercoiling to respond to
signi®cant changes in supercoiling (cf. below). Therefore,
the lack of control of topoisomerase I on gyrase expression
must again have been due to, rather than caused by, the
small effect the former had on DNA supercoiling. Clearly
Fig. 3. DNA gyrase expression as a function of aLk. T he concentration
of D NA gyrase is plotted at dierent aLk values obtained by incu-
bation of strain HWA36 with dierent concentrations of IPTG. Data
from ®ve independent experiments are shown using dierent symbols
for each. Data points are averages of three me asurements. The
error bars denote the standa rd error of the mean. Wild-type is
shown as a closed circle. The elasticity coecient de®ned as
e
kt
gyrase
supercoiling

dkt
gyrase
daLk
Á
aLk
kt
gyrase
was calculated by multiplying the derivative
of the ®tted curve at each point of the graph with the quotient of the
respective x/y coordinates. At wild-type level of supercoiling a n elasti-
city coecient of )1.6 was calculated.
Ó FEBS 2002 Control of DNA supercoiling by topoisomerase I (Eur. J. Biochem. 269) 1665

Discussion we shall address these possibilities in detail.
DISCUSSION
Homeostatic control of DNA supercoiling in prokaryotes
has been proposed previously [6]: the enzyme that causes
negative supercoiling, i.e. DNA gyrase, was repre ssed b y
highly negative supercoiling. This observation showed
homeostatic control of DNA gyrase expression but not of
DNA supercoiling itself, as the implications for DNA
supercoiling were not determined. In addition the strength
of the homeostatic control and whether it also occurred i n
and around the physiological state, had not yet been
addressed.
DNA gyrase and topoisomerase I are considered to be
the m ost important en zymes in c ontrolling t he level of
supercoiling in E. coli [23]. This suggests two mechanisms of
homeostasis [6,9]. One is that decreased supercoiling may
enhance the expression level of DNA gyrase that then leads
to an increase of supercoiling. The second is that the
decreased supercoiling diminishes the expression level of
topoisomerase I, which leads to enhanced supercoiling.
There should be two additional, more direct mechanisms.
One consists of the phenomenon that the rate at which
DNA gyrase supercoils DNA may decrease with the extent
to which that DNA is supercoiled, with zero activity at t he
static head situation [24]. The other relies on a more than
proportional dependence of the catalytic rate of topoisom-
erase I on the extent of DNA supercoiling. Homeostasis of
DNA supercoiling could be called ÔsubtleÕ if all four of these
mechanisms were involved. It could be called ÔsimpleÕ if only
one mechanism was operative. In our analysis we focus on

max
of the topoisom-
erase I reaction. Note that the lower case c is used for this
type of control coef®cient. The v alue of the c ontrol
coef®cient is equal to the percentage change that is observed
in the aLk upon a percentage change in the activity of
topoisomerase I.
In addition gyrase activity will in¯uence DNA supercoil-
ing. The sensitivities (de®ned as elasticity coef®cients by
metabolic control analysis) of both enzymes to changes in
supercoiling will determine the magnitude of the control
coef®cients. Using the concentration summation and
connectivity theorems (cf. [22]). th e intrinsic control by
Fig. 4. Topoisomerase I expression as a function of aL k. The concen-
tration o f topoisomerase I is plotted at dierent aLk values ob tain ed
by incubation of strain PJ4273 [11] with dierent concentrations
of IPTG. Data from two in depen dent experiments are s hown using
dierent symbols f or each. Data p oints are averages of three
measurements. The error bars denote the standard error of the mean.
Wild-type is sho wn as a closed circle. The ela sticity coe cient d e®ned
as e
kt
topoisomerase I
supercoiling

dkt
topoisomerase I
daLk
Á
aLk

kt
topoisomerase I
supercoiling
and e
kt
gyrase
supercoiling
, re¯ecting
the s ensitivity of transcription of topoisomerase I and DNA
gyrase for D NA superco iling. Certain sim pli®cations were
made in [25], i.e. grouping of transcription and translation,
assuming that transcription/translation i s product insensit-
ive a nd mRNA and p rotein degradation follow ®rst order
kinetics (for a more general treatment, see [26]). Expressing
the control coef®cients in terms of elasticities in such a
system leads to the following expression for the ÔglobalÕ
control (hence the capital Cs) by the topoisomerases on
supercoiling:
C
supercoiling
gyrase

1
e
v
topoisomerase I
supercoiling
e
kt
topoisomerase I

H  1À
dlnaLk
dlnv
topoisomerase I

system at steady state
 1 ÀC
supercoiling
topoisomer ase I
With this de®nition, when a 10% increase in relaxation
activity leads to a 10% decrease in linking number, no
relaxation is prevented and H becomes equal to 0; there is no
homeostasis. When there is no decrease in linking number,
H equals 1, i.e. there is complete homeostasis. The utility of
the d e®nition is that we can now evaluate intermediary cases
between no and complete homeostasis. Homeostasis of
DNA supercoiling in E. coli is such an intermediary case: in
terms of t his de®nition, it is quanti®ed as 1 ) 0.13  0.87,
i.e. 87% of complete homeostasis. This shows that home-
ostasis of DNA supercoiling is quite strong.
From Eqn (3) and the de®nition of H, it follows that this
coef®cient is independent of whether topoisomerase I is
activated or DNA gyrase i s inhibited to compromise DNA,
and equal to:
H
supercoiling

e
v
topoisomerase I

4
The elasticities o f gyrase a ctivity and gyrase expre ssion for
supercoiling are ne gative (i.e. the activity and expression of
DNA gyrase i s inhibited not stimulated by higher levels of
supercoiling) and those of topoisomerase I positive. Con-
sequently all four of these elasticities c an contribute
positively to the homeostatic control of supercoiling. The
equation suggests that the subtlety of the homeostatic
control in the above sense can be determined by inspecting
whether all four elasticities are of signi®cant magnitudes.
In the strain used to manipulate the topoisomerase I
concentration, expression of topoisomerase I is controlled
by IPTG and the elasticity with respect to supercoiling is
zero. The transcription rate of topoisomerase I was modu-
lated and the effect on topoisomerase I concentration and
supercoiling measured, leading to a measured value for the
inherent control coef®cient of topoisomerase I with r espect
to DNA supercoiling (in metabolic control analysis terms, a
coresponse coef®cient):
t
topoisomerase I
O
supercoiling
e
topoisomerase I

C
supercoiling
t
topoisomerase I

t
gyrase
C
e
gyrase
t
gyrase

1
e
v
topoisomerase I
supercoiling
e
kt
topoisomerase I
supercoilin g
Àe
v
gyrase
supercoiling
6
For topoisomerase I an inherent control of )0.14 ( 0.03)
was determined experimentally while for DNA gyrase an
inherent control of 0.17 ( 0.01) was measured [11]. Global
control coef®cients (Eqn 3) can be calculated from the
inherent control coef®cients by adding the elasticities of
expression of DNA gyrase and topoisomerase I (i.e. )1.6
and +0.56, respectively) for supercoiling in Eqns (6) a nd (5),
respectively. In this manner a global control of supercoiling

gyrase
supercoiling
À
1
t
topoisomerase I
O
supercoiling
e
topoisomerase I
À1X6  7X2  5X6
And from Eqn (6):
e
v
topoisomerase I
supercoiling
À e
v
gyrase
supercoiling
Àe
kt
topoisomerase I
supercoiling

1
t
topoisomerase I
O
supercoiling

of DNA gyrase or topoisomerase I was measured using
promoter fusion, always large perturbations in DNA
supercoiling were made [9,27]. As can been seen from
Figs 3 and 4 the sensitivity of gene expre ssion to s upercoil-
ing does depend on th e level of supercoiling, especially for
the topoisomerase I, which is almost insensitive at wild-type
levels of supercoiling and much more sensitive at h igh levels
of supercoiling. One can compare the results of these earlier
studies with ours by extrapolating our results to larger
changes in supercoiling. Fusion of the gyrB promoter to the
galactokinase gene showed a two to three f old increase upon
inhibition of gyrase with coumermycin [27]. Our results are
in good agreement with this: Extrapolation of our ®ts i n
Fig. 3 to an aLk of 0 (corresponding to coumermycin
inhibition) indicates a 2.8-fold induction. Fusion of the topA
promoters to the galactokinase gene showed a twofold to
fourfold inhibition of expression upon addition of gyrase
inhibitors [9]. With our DNA gyrase modulatable strain we
did not observe a strong effect upon decreasing the level of
supercoiling below the wild-type level. Rather at higher
levels of supercoiling an i nduction of topoisomerase I was
observed. Perhaps t opoisomerase I expression becomes
more sensitive for supercoiling when the DNA relaxes
more than was tested in our strains. In the e arlier studies the
promoter fusions were plasmid c onstructs while in the
present study we looked at the native chromosomal
promoter activities. The location of the promoter might
very well have an effect on its sensitivity for supercoiling.
We have shown that for the speci®c case of DNA
supercoiling, homeostatic control resides predominantly

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