Proc. Natl. Acad. Sci.
USA
Vol. 94, pp. 7263–7268, July 1997
Biochemistry
A model of excitation and adaptation in bacterial chemotaxis
PETER A. SPIRO*, JOHN S. PARKINSON†, AND HANS G. OTHMER*‡
Departments of *Mathematics and †Biology, University of Utah, Salt Lake City, UT 84112
Communicated by Charles S. Peskin, New York University, Hartsdale, NY, April 24, 1997 (received for review August 26, 1996)
ABSTRACT Bacterial chemotaxis is widely studied because
of its accessibility and because it incorporates processes that are
important in a number of sensory systems: signal transduction,
excitation, adaptation, and a change in behavior, all in response
to stimuli. Quantitative data on the change in behavior are
available for this system, and the major biochemical steps in the
signal transductionyprocessing pathway have been identified.
We have incorporated recent biochemical data into a mathemat-
ical model that can reproduce many of the major features of the
intracellular response, including the change in the level of
chemotactic proteins to step and ramp stimuli such as those used
in experimental protocols. The interaction of the chemotactic
proteins with the motor is not modeled, but we can estimate the
degree of cooperativity needed to produce the observed gain
under the assumption that the chemotactic proteins interact
directly with the motor proteins.
Chemotaxis (or more accurately, chemokinesis), the process by
which a cell alters its speed or frequency of turning in response
to an extracellular chemical signal, has been most thoroughly
studied in the peritrichous bacterium Escherichia coli. E. coli
exhibits sophisticated responses to many beneficial or harmful
chemicals. In isotropic environments the cell swims about in a
random walk produced by alternating episodes of counterclock-
wise (CCW) and clockwise (CW) flagellar rotation. CCW rota-
tion pushes the cell forward in a fairly straight ‘‘run,’’ CW rotation
triggers a random ‘‘tumble’’ that reorients the cell. The durations
of both runs and tumbles are exponentially distributed, with
means of 1.0 s and 0.1 s, respectively (1). In a chemoeffector
gradient, the cell carries out chemotactic migration by extending
runs that happen to carry it in favorable directions. Using specific
chemoreceptors to monitor its chemical environment, E. coli
perceives spatial gradients as temporal changes in attractant or
repellent concentration. The cell in effect compares its environ-
ment during the past second with the previous 3–4 s and responds
accordingly. Attractant increases and repellent decreases tran-
siently raise the probability of CCW rotation, or ‘‘bias,’’ and then
a sensory adaptation process returns the bias to baseline, enabling
the cell to detect and respond to further concentration changes.
The response to a small step change in chemoeffector concen-
tration in a spatially uniform environment occurs over a 2- to 4-s
time span (2). Saturating changes in chemoeffector concentration
can increase the response time to several minutes (3).
Many bacterial chemoreceptors belong to a family of trans-
membrane methyl-accepting chemotaxis proteins (MCPs) (re-
viewed in ref. 4). Among the best-studied MCPs is Tar, the E.
coli receptor for the attractant aspartate. Tar has a periplasmic
binding domain and a cytoplasmic signaling domain that
communicates with the flagellar motors via a phosphorelay
sequence involving the CheA, CheY, and CheZ proteins (see
Fig. 1). CheA, a histidine kinase, first autophosphorylates and
then transfers its phosphoryl group to CheY.
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© 1997 by The National Academy of Sciences 0027-8424y97y947263-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
7263
Phospho–CheY interacts with switch proteins in the flagellar
motors to augment CW rotation, CCW being the default state in
the absence of Phospho–CheY. CheZ assists in dissipating CW
signals by enhancing the dephosphorylation of CheY. Tar con-
trols the flux of phosphate through this circuit by forming a stable
ternary complex (TaryCheWyCheA) that modulates CheA au-
tophosphorylation in response to changes in ligand occupancy or
methylation state. CheA phosphorylates more slowly when the
receptor is occupied than when it is not.
Changes in MCP methylation state are responsible for sensory
adaptation. Tar has four residues that are reversibly methylated
by a methyltransferase, CheR, and demethylated by a methyles-
terase, CheB. CheR activity is unregulated, whereas CheB, like
CheY, is activated by phosphorylation via CheA. Thus, receptor
methylation level is regulated by feedback signals from the
signaling complex, which can probably shift between two confor-
mational states having different rates of CheA autophosphory-
lation. Attractant binding and demethylation shift the equilibrium
toward a low CheA activity state; attractant release and methyl-
ation shift the equilibrium toward a high CheA activity state. A
receptor complex that is bound to attractant but not highly
methylated can be thought of as a ‘‘sequestered’’ state (5) which
is unable to autophosphorylate at a significant rate.
E. coli can sense and adapt to ligand concentrations that
range over five orders of magnitude (5). In addition, the
machinery for detection and transduction is exquisitely sensi-
tive to chemical stimuli. The cell can respond to exponential
increases (‘‘ramps’’) in attractant levels that correspond to
rates of change in fractional occupancy of only 0.1% per
second. Consequently, the cell can respond even if only a small
fraction of its receptors have changed occupancy state during
a typical sampling period.
These experimental observations raise several questions. First,
how does the cell achieve the extreme sensitivity that is observed?
Second, why are there multiple methylation states of the recep-
tor? In this paper we describe a mathematical model based on
known kinetic properties of Tar and the phosphorelay signaling
components that accounts for this exquisite sensitivity. In the
model, excitation results from the reduction in the autophos-
phorylation rate of CheA when Tar is bound to a ligand, and
adaptation arises from methylation of the receptor. The disparity
in the time scales of these processes produces a ‘‘derivative-’’ or
‘‘temporal-sensing’’ mechanism with respect to the ligand con-
centration. The model makes essential use of the multiple meth-
ylation states to achieve adaptation, and can be used to derive
quantitative relations between certain key rates and the behav-
ioral responses to experimental protocols, and to predict the
cooperativity needed to achieve the observed gain.
A Qualitative Description of the Response
to Different Stimuli
We assume that Tar is the only receptor type, that the Tar–
CheA–CheW complex does not dissociate, and that Tar, CheA,
and CheW are found only in this complex. We also assume that
methylation of the multiple sites occurs in a specified order (6, 7).
Abbreviations: CCW, counterclockwise; CW, clockwise; MCP, meth-
yl-accepting chemotaxis protein.
‡To whom reprint requests should be addressed.
7264 Biochemistry: Spiro et al.
FIG. 1. Signaling components and pathways for E. coli chemotaxis.
Chemoreceptors (MCPs) span the cytoplasmic membrane (hatched
lines), with a ligand-binding domain deployed on the periplasmic side
and a signaling domain on the cytoplasmic side. The cytoplasmic Che
signaling proteins are identified by single letters—e.g., A 5 CheA.
MCPs form stable ternary complexes with the CheA and CheW
proteins to generate signals that control the direction of rotation of the
flagellar motors. The signaling currency is in the form of phosphoryl
groups (;P), made available to the CheY and CheB effector proteins
through autophosphorylation of CheA. CheYp initiates flagellar re-
sponses by interacting with the motor to enhance the probability of
clockwise rotation. CheBp is part of a sensory adaptation circuit that
terminates motor responses. MCP complexes have two alternative
signaling states. In the attractant-bound form, the receptor inhibits
CheA autokinase activity; in the unliganded form, the receptor
stimulates CheA activity. The overall flux of phosphoryl groups to
CheB and CheY reflects the proportion of signaling complexes in the
inhibited and stimulated states. Changes in attractant concentration
shift this distribution, triggering a flagellar response. The ensuing
changes in CheB phosphorylation state alter its methylesterase activ-
ity, producing a net change in MCP methylation state that cancels the
stimulus signal (see ref. 4 for a review).
Our primary objective is to model the response to attractant,
which probably involves only increases in the average methylation
level above the unstimulated level of about 1.5–2 methyl esters per
transducer (8). For this reason and for simplicity, we consider only
the three highest methylation states of Tar. In the model we
postulate that only the phosphorylated form of CheB (CheBp)
has demethylation activity. We assume that the phosphorylation
state of CheA does not affect the ligand-binding reactions of the
receptor with which it forms a complex, and does not affect the
activities of CheR or CheBp. We further assume that the forma-
tion of a complex between Tar and CheR does not affect the
ligand-binding properties of Tar, and that it does not affect the
autophosphorylation rate of the attached CheA until the transi-
tion to the more highly methylated state (and dissociation of the
complex) occurs. We assume that CheZ activity is unmodulated,
although it has been suggested (9) that an as-yet-unidentified
mechanism for modulating CheZ activity could account for the
observed gain. Later we consider the case of modulated CheZ
activity. Finally, we assume that the rates of the phosphotransfer
reactions between CheA and CheY or CheB are not affected by
the occupancy or methylation state of the receptor.
In Fig. 2 we illustrate the receptor states and the network of
transitions between them that are used in the model. Details
of the phosphotransfer reactions are shown in Fig. 3, together
with the dephosphorylation reactions for CheB and CheY. The
details of the reactions in these figures are given in Table 1.
Before we introduce the equations we present a qualitative
description of how the system works.
First consider the response of the network to a step increase
in attractant. The ligand-binding reactions are the fastest, so
the first component of the response is a shift in the distribution
of states toward the ligand-bound states (i.e., from states at the
top to those at the bottom in Fig. 2). This increases the fraction
of receptors in the sequestered states (LT2, LT2p, and possibly
LT3, LT3p), and because these states have the same phospho-
transfer rates but much lower autophosphorylation rates than
the corresponding ligand-free states, the distribution subse-
Proc. Natl. Acad. Sci. USA 94 (1997)
FIG. 2. The ligand-binding, phosphorylation, and methylation reac-
tions of the Tar–CheA–CheW complex, denoted by T. LT indicates a
ligand-bound complex. Vertical transitions involve ligand binding and
release, horizontal transitions involve methylation and demethylation,
and front-to-rear transitions involve phosphorylation and the reverse
involve dephosphorylation. The details of the phosphotransfer steps are
depicted in Fig. 3. Numerical subscripts indicate the number of methyl-
ated sites on Tar, and a subscript p indicates that CheA is phosphorylated.
The rates for the labeled reaction pairs are given in Table 3.
quently shifts toward states containing unphosphorylated
CheA. This in turn leads to a reduction in CheYp, a decreased
rate of tumbling, and an increase in the run length. This
constitutes the excitation response of the system.
Next methylation and demethylation, which are the slowest
reactions, begin to exert an effect. CheR methylates ligand-bound
receptors more rapidly than unbound receptors, and the decrease
in CheAp that results from excitation causes a decrease in the level
of CheBp, thereby reducing the rate of demethylation. As a result,
the distribution shifts toward higher methylation states. However,
autophosphorylation of CheA is faster, the higher the methyl-
ation state of the Tar–CheA–CheW complex, and therefore in the
last phase of the response there is a shift toward the states
containing CheAp via transitions along the right face of the
network. Consequently, the net effect of an increase in attractant
is to shift the distribution of receptor states toward those which
are ligand-bound and more highly methylated, but the total level
of receptor complex containing CheAp (the sum of the states at
the rear face of the network) returns to baseline. As a result, the
total phosphotransfer rate from CheAp species to CheY returns
to the prestimulus level, which means that CheYp returns to its
prestimulus level. Under the assumption that only CheYp and
CheY interact with the motor complex, this in turn implies that
the bias returns to its prestimulus level. Thus the cell can respond
to an increase in ligand by a transient decrease in tumbling, but
it adapts to a constant background level of ligand and retains
sensitivity to further changes in the ligand concentration. Of
course this qualitative description must be supplemented by
numerical results which demonstrate that the model can also
produce quantitatively correct results using experimentally based
rate coefficients. This is done in the following section.
FIG. 3. Detail of the phosphotransfer reactions corresponding to
the labels 8–13 in Fig. 2.
 Biochemistry: Spiro et al. Proc. Natl. Acad. Sci. USA 94 (1997) 7265

Table 1. Details of reactions depicted in Figs. 2 and 3 dB p

5 k b P ~ B 0 2 B p! 2 k 2bB p, [3]
Reaction dt
Description labels Kinetics and rate labels
k1 where P [ T2 p 1 LT2 p 1 T3p 1 LT3p 1 T4p 1 LT4p is the total
Methylation (first order 1–4 T2 1 R 3 T3 1 R
amount of phosphorylated receptor complex. In addition, the
kinetics) k1b k1c total amounts of T, Y, B, and R are conserved. These four
Methylation (Michaelis– 1–4 T2 1 R º T2 R 3 T3 1 R
Menten kinetics)
k1a conditions can be used to eliminate four variables, which was
k21 done for Y and B in Eqs. 2 and 3, and the resulting system can be
Demethylation 1–4 T3 1 Bp 3 T2 1 Bp
k5 integrated numerically, using the parameter values given in the
Ligand binding 5–7 T2 1 L º LT2 tables. As we remarked earlier, we assume that the motor
k25
k8 interacts only with CheYp and possibly CheY, and in view of the
Autophosphorylation 8–13 T2 3 T2p
kb small number of motor complexes in a cell, we further assume that
Phosphotransfer 8–13 T2p 1 B 3 T2 1 Bp the motor does not affect the budgets of these species. It then
ky
T2p 1 Y 3 T2 1 Yp follows from the conservation of Y that perfect adaptation of the
k2b
Dephosphorylation Bp 3 B bias only requires perfect adaptation of CheYp.
k2y The responses obtained from this three-methylation-state
Yp 1 Z 3 Y 1 Z model are shown in Fig. 4A–C and the concentrations and
rates used are listed and compared with measured values in
In ref. 10 we examine the response of a simplified model that Tables 2 and 3. The system exhibits both a significant response
incorporates just two methylation states to step changes and to to a slow ramp and an appropriate adaptation time for a small
a slow exponential ramp. The results of this analysis impose step. It also exhibits the correct adaptation time to a saturating
different conditions on the methylation and demethylation step in ligand concentration.
rates, because a significant ramp response results only when
Analysis of the Gain
these rates are considerably slower than those which reproduce
observed adaptation times to small steps. To capture both of In the preceding figures we have chosen the step size so that
these responses a model must contain at least three methyl- the maximum deviations of the CheYp concentration from
ation states, and the transitions between the lowest and baseline are equal in Fig. 4A and B, and thus the maximum
intermediate methylation states must be considerably faster change in bias in response to the corresponding ramp (0.015
than the transitions between the intermediate and highest s21) and step (11% change in receptor occupancy) will be the
states. In such a scheme the adaptation time to small steps is same. Experimentally it is found that the maximum change in
controlled by the transitions between the doubly and triply
bias in response to this ramp is about 0.3 (figure 4A in ref. 16),
methylated states in Fig. 2, and the ramp response is controlled
and so our model, coupled with a scheme for the interaction
by the slower transitions between the triply and fully methyl-
of CheYp with the motor, would produce a maximum gain of
ated states. The three-methylation-state model described in
about 0.3y0.11 5 2.73 in response to the step used in Fig. 4B.
the following section also captures the correct time scale for
the adaptation response to saturating levels of attractant. This is consistent with the finding (17) that the maximum gain
is about 6, though not with the much higher gain of 55 reported
A Three-State Model Captures the Ramp, Step, and (18). The model thus demonstrates that ramp experiment
Saturation Responses results are consistent with the lower estimates regarding the
gain for small steps. However the source of the gain remains
A minimal model must have three methylation states, and we undetermined, because even though the deviations of CheYp
assume faster methylation and demethylation rates for transitions concentration from baseline are significant ('9%), they are
between the two lowest methylation states, and slower transition
small compared with the reported change (0.3) in bias for this
rates between the two highest methylation states. We use Michae-
ramp stimulus (16). Thus one or more mechanisms to amplify
lis–Menten methylation kinetics, since CheR activity is thought to
the internal CheYp signal must be involved, and the major
be saturated under normal conditions (11). This precludes an
possibilities are cooperative effects in the interaction of CheY
analytical derivation of the relationships between kinetic param-
eters which guarantee perfect adaptation, by which we mean that species with the motor and modulation of CheZ activity.
the bias returns precisely to baseline in the face of any constant To explore these possibilities, we define the gain as
attractant level below saturation, (10). Instead we have tuned the db
rate constants of the full system by trial and error so that it adapts g52 , [4]
well over a large range of ligand concentrations. d ln p
The mathematical description of the model is based on mass
action kinetics for all the steps except methylation. Because the where b is the bias, or probability that a flagellum will be
equations that govern the evolution of the amounts in the various rotating CCW, and p [ ky P is the pseudo-first order rate
states of the receptor complex are similar and easy to derive, we constant for phosphorylation of CheY. We have chosen this
only display one of them, and we indicate the process contributing definition of gain for mathematical convenience, but it can be
to the rate of change beneath the corresponding rate expression. shown (10) that for small steps this definition of g is consistent
with the definition of gain given in ref. 17, namely, the change
dT2 2Lk
« 5T2 1 k25LT2 k
«8T2 Bpk 2 1T3 in bias per percent change in receptor occupancy.
5 2 1 «
dt Ligand bindingyrelease Phosphorylation Demethylation When the phosphorylation reactions are at pseudo-
T2 equilibrium, one can show that
kyT2p~Y0 2 Yp! 1 kbT2p~B0 2 Bp! Vmax K 1 T
1 «2 « R 2
p
Phosphotransfer methylation [1]
y5 , [5]
p1z
We write the equations for CheYp and CheBp as
where y [ YpyY0 [ [0,1] is the dimensionless amount of CheY
dY p in phosphorylated form, and p and z [ k-y Z are the production
5 k y P ~ Y 0 2 Y p! 2 k 2y ZY p, [2]
dt and loss coefficients, respectively, of y. We note that
7266 Biochemistry: Spiro et al. Proc. Natl. Acad. Sci. USA 94 (1997)

FIG. 4. Response of the system depicted in Fig. 2 to various chemoattractant (aspartate) stimulus protocols: a ramp of rate 0.015 s21 (Left), a
step at t 5 10 s from zero concentration to a concentration (0.11 mM) that is 11% of the Kd for ligand binding (Center), and a step at t 5 100 s
from zero concentration to a concentration (1 mM) that is 1,000 times the Kd for ligand binding (Right). Time series in the top row of figures are
for Che Yp (solid line), CheBp (short dashes), and aspartate (long dashes). The concentrations and rates used are listed and compared to measured
values in Tables 2 and 3. The maximum change in [Che Yp] from baseline for both the ramp and small step responses is 9%. Time series in the
bottom row are the corresponding bias responses, using a Hill coefficient of 11 for binding of both Che Yp and the competitive inhibitor Che Y
to the motor. (Compare the left trace to figure 4A in ref. 12; the center trace to figure 4 in ref. 2, figure 2 in ref. 14, and figure 8 in ref. 13; and
the right trace to figure 8 in ref.3.

­ ln y z via cooperative binding, or possibly via some cooperative

5 , 1.
­ ln p p 1 z
Thus without modulation of CheZ activity, the fractional change
in CheYp levels must be less than the change in receptor
occupancy, so that no signal amplification is possible upstream of
the interaction of CheY and CheYp with the motor.
To examine the effect on the gain of cooperative interactions
of CheY species at the motor and modulation of CheZ activity,
we expand Eq. 4, using Eq. 5, to obtain
[6] interaction among subunits of the switch which bind individ-
ually to CheY molecules. Here we consider the first possibility
(the second is treated in ref. 10), and we assume that a motor
unit binds either Che Y or Che Yp according to
jY p 1 M º M ~ Y p! j, [8]
kY 1 M º M ~ Y ! k. [9]
Here j and k are the number of molecules of CheYp and CheY,

S D
respectively, which bind to the motor, M represents motor
db d ln z unbound to either CheY species, M(Yp)j represents motor in
g5 2 y~1 2 y! 12 5 v~1 1 w!. [7]
dy d ln p complex with Yp , and M(Y)k represents motor in complex with
Y. We assume that the binding reactions Eqs. 8 and 9 equili-
Here v [ 2y(1 2 y)dbydy is the gain due only to cooperative brate rapidly, and study only the steady-state quantities
interaction of CheY with the motor, and w [ 2dlnzydlnp is,
for small steps at least, the fractional change in CheZ activity M 1
m; 5 , [10]
per fractional change in receptor occupancy (10). We have M 0 1 1 A j y 1 A k~ 1 2 y ! k
j

assumed here that CheZ does not interact directly with the
motor (19), and acts only by dephosphorylating CheY. This M ~ Y p! j Ajyj
mj ; 5 , [11]
expression makes it clear that the two possible sources of M0 1 1 A j y j 1 A k~ 1 2 y ! k
cooperativity act multiplicatively.
To determine the gain achievable, consider first the factor M~Y!k A k~ 1 2 y ! k
mk ; 5 . [12]
v. We assume that CheY species interact with the motor by M0 1 1 A j y j 1 A k~ 1 2 y ! k
binding to the flagellar switch, and we allow for the possibility
that both CheYp and unphosphorylated CheY may bind (19). Here M0 is the total concentration of motor, and Aj and Ak are
Interaction with the motor may involve cooperativity, either the ratios of the association and dissociation constants for
CheYp and unphosphorylated CheY, respectively.
Table 2. Conserved quantities used in the model (see ref. 3) We assume that the unbound motor M exists in CCW mode
Species T R B Y Z with probability one (19), and that the Mj and Mk states have
probabilities fj and fk, respectively, of being in CCW mode.
Concentration, mM 8 0.3 1.7 20 40
Then the bias is given by
 Biochemistry: Spiro et al. Proc. Natl. Acad. Sci. USA 94 (1997) 7267

Table 3. Rates used in the model, and corresponding values from the literature
Reaction Rate constant Value Literature value Ref.
T 2R 3 T 3 1 R k 1c 0.17 s21 0.17 s21 12
T 3R 3 T 4 1 R k 2c 0.1k 1c .0.02k 1c* 13
LT 2R 3 LT 3 1 R k 3c 30k 1c 15k 1c 2 30k 1c† 13
LT 3R 3 LT 4 1 R k 4c 30k 2c 15k 2c 2 30k 2c† 13
T n 1 R º T nR k 1byk 1a, . . . , k 4byk 4a 1.7 mM 1.7 mM 12
T3 1 Bp 3 T2 1 Bp k 21 4 3 10 5 M21zs21 3 3 10 4 M21zs21 11
T4 1 Bp 3 T3 1 Bp k 22 3 3 10 4 M21zs21 .3.0k 21‡ 13
LT 3 1 B p 3 LT 2 1 B p k 23 k 21 k 21§ 13
LT 4 1 B p 3 LT 3 1 B p k 24 k 22 k 22§ 13
L 1 T 3 LT k5, k 6, k 7 7 3 10 7 M21zs21 7 3 10 7 M21zs21 3¶
LT 3 L 1 T k 25, k 26, k 27 70 s21 70 s21 3¶
T 2 3 T 2p k8 15 s21 17 s21 14
T 3 3 T 3p k9 3k 8
T 4 3 T 4p k 10 3.2k 8
LT 2 3 LT 2p k 11 0
LT 3 3 LT 3p k 12 1.1k 8
LT 4 3 LT 4p k 13 0.72k 10 k 10 15
B 1 T np 3 B p 1 T n kb 8 3 10 5 M21zs21 8 3 10 5 M21zs21 11i
Y 1 T np 3 Y p 1 T n ky 3 3 10 7 M21zs21 3 3 10 7 M21zs21 14
Bp 3 B k 2b 0.35 s21 0.35 s21 14
Yp 1 Z 3 Y 1 Z k 2y 5 3 10 5 M21zs21 5 3 10 5 M21zs21 14
*Methylation rates of different methylation sites vary by a factor of up to 50.
†Ligand binding increases methylation rates of different methylation sites by a factor of 15–30.
‡Demethylation rates of different methylation sites vary by a factor of up to 3.
§Ligand binding has little effect on demethylation rate.
£Estimated from figure 10 in ref. 3.
i
Estimated from figure 3 in ref. 11.

1 1 f j A j y j 1 f k A k~ 1 2 y ! k g
b 5 m 1 f jm j 1 f km k 5 .[13] w$4 2 1. [18]
1 1 A j y j 1 A k~ 1 2 y ! k n

One can show that the optimal values of fj and fk are 0 and 1, Thus, without cooperative effects at the switch, we must have
respectively (10), and using these v is given by w $ 10, and for moderate cooperativity at the switch, for
example n 5 6 (20), we must have w $ 0.8. A gain of 6 (17)
v ~ y# ! 5
1
4
Sj ~ 1 2 y# ! 1 ky#
ak
1 1 ak
. D [14]
requires w $ 23 and w $ 3 in these respective cases.
Finally, we note that an additional cooperative step probably
occurs in the interactions between flagella, since the bias of an
where ak 5 Ak(1 2 y)k and y is the baseline CheYp concen- individual flagellum in the absence of stimulation [.0.64 (2)]
tration. Because this expression is monotonically increasing in is less than that of a swimming cell [.0.9, assuming mean run
ak, v(y# ) is maximized with respect to ak when ak is as large as and tumble durations of 1.1 s and 0.14 s, respectively (21)]. We
possible, indicating that both CheY species should have strong can estimate what the threshold number of flagella might be
for this cooperative interaction if we adopt the ‘‘voting hy-
binding affinities for the flagellar switch. In the limit as ak 3
pothesis’’ (21, 22), whereby the biases of the individual flagella
`, Eq. 14 becomes
are identical and independent, and the probability that the
1 flagella will form a bundle is one when the number of flagella
v ~ y# ! 5 ~ j ~ 1 2 y# ! 1 ky# ! . [15] turning CCW equals or exceeds a threshold u and zero
4 otherwise. Then the bias B of the cell is given by
Finally, maximizing Eq. 15 with respect to y# (subject to y# [ [0, 1])
OS D
N
shows that the maximum possible gain at the flagellar switch is N j
B5 b ~ 1 2 b ! N2j, [19]
j
j5u
1
v ~ y# ! 5 n, n ; max~ j, k ! . [16] N
4 (not B 5 ( j5u b j(1 2 b) N2j, as claimed by Weis and Koshland
(22)], where N is the total number of flagella and b is the bias
Thus, a gain of 2.7 requires a Hill coefficient of at least 11 if of an individual flagellum. For b 5 0.64 and either n 5 6 or
the only cooperativity is in the interaction of CheY species with n 5 8 (23), we find that B . 0.9 when u 5 Ny2, and thus a
the switch. In Fig. 4 D–F we show the bias resulting from the simple majority rules.
CheYp response in Fig. 4 A–C using a Hill coefficient of 11 for
Discussion
both CheY species (j 5 k 5 11).
The full expression for the gain now becomes In our model of aspartate signal transduction via Tar, excitation
is the result of the reduced autophosphorylation rate of the
1 ligand-bound state of the receptor, which reduces the level of
g# n~1 1 w!, [17]
4 CheAp and CheYp, thereby reducing the tumbling rate. Adapta-
tion results from the enhanced rate of methylation of bound states
and so the fractional change in CheZ activity relative to the and the fact that methylation increases the rate of autophosphor-
fractional change in receptor occupancy is ylation, which returns CheAp and CheYp to their prestimulus
7268 Biochemistry: Spiro et al.
levels. The results we present demonstrate that the model can
reproduce the experimentally observed responses to both step
increases and slow ramps using experimentally determined values
for most of the parameters. The disparity between the time scale
of excitation, which is fast, and that of adaptation, which is slow,
implies that the transduction system can function as a ‘‘derivative
sensor’’ with respect to the ligand concentration: the DC com-
ponent of a signal is ultimately ignored if it is not too large. This
provides a bacterium with a temporal sensing mechanism without
the need for any type of memory beyond that embodied in the
disparity in time scales between excitation and adaptation.
In ref. 10 we show that, as is seen experimentally (16), the
magnitude of the response to a slow ramp is an increasing
function of ramp rate, and so we may understand the ramp
response as the result of a difference between the rate at which
receptors enter the sequestered state via increases in receptor
occupancy, and the rate at which they exit via transitions between
methylation states. Steeper ramps result in larger differences
between the entrance and exit rates, and thus in larger responses.
The threshold ramp response (16) occurs when these rates are
equal. In response to a ramp, the deviation from baseline of the
level of sequestered receptor is an approximately linear function
of ramp rate. If the downstream steps in the transduction pathway
operate in a linear range, then the bias response will in turn be
approximately linear in the ramp rate, consistent with the finding
of Block et al. (16).
Three methylation states were necessary to accurately repro-
duce the responses to both step and ramp stimuli, because these
responses place competing restrictions on the effective methyl-
ation and demethylation rates. For step stimuli given at a baseline
attractant concentration of zero (17, 18), adaptation is dominated
by the fast transitions between the two lowest methylation states,
which provide a sufficiently fast adaptation response. At the
higher attractant concentrations of ramp experiments (16, 18),
receptors are on average more highly methylated, and so the
slower transitions between the two highest methylation states are
more prominent, providing a significant ramp response.
The sensitivity, or gain, of the signal transduction system is
found experimentally to be quite high, but the source of this
sensitivity is unknown. We have shown that the sensitivity ob-
served in response to ramp stimuli (16, 18) is consistent with the
moderate estimate of 6 for the maximum gain of the system (17)
obtained from step experiments, but that cooperativity equivalent
to a Hill coefficient on the order of 11 is necessary to produce the
desired gain. There are several potential sources of cooperativity.
Binding of CheYp to the flagellar switch is thought to be
cooperative (20), and we have shown that competitive inhibition
by unphosphorylated CheY can enhance the sensitivity. Because
the motor contains 26 or 27 subunits of switch protein FliF (19),
it is conceivable that high cooperativity occurs either in binding
to the switch or else in interactions among switch subunits bound
individually to CheYp. CheZ phosphatase activity may also be
modulated in a manner that exhibits cooperativity. The system
gain could be significant if CheZ activity were positively corre-
lated with the level of sequestered CheA, because small fractional
changes in receptor occupancy can correspond to large fractional
changes in sequestered CheA. As an example, the cells possess a
short form of CheA (CheAs) which may bind several molecules
of CheZ (24). If it does so when the associated transducer is
unbound to attractant or highly methylated, and releases these
molecules into the cytoplasm upon attractant binding, the effec-
tive concentration of CheZ will be raised following the latter
event. Alternatively, the CheAs–CheZ complex may amplify
CheZ phosphatase activity [Wang (1996) cited in ref. 25], which
could produce high gain if the complex were to form upon
attractant binding. Polymerization of CheZ in the presence of
CheYp is another potential mechanism for signal amplification,
though it is more likely that this reaction instead enhances the
adaptation response (25). A further source of gain might be found
in cooperative interactions among receptors in close proximity to
Proc. Natl. Acad. Sci. USA 94 (1997)
one another, a possibility raised by the existence of a ‘nose spot’
of elevated receptor density (26).
We have shown that the effects of cooperativity at different
locations in the signal transduction pathway are likely to be
multiplicative, and thus the total system gain may be the result
of moderate cooperativity occurring at two or more of the
above-mentioned locations. For example, a Hill coefficient of
6 at the flagellar switch (20) and a moderate degree of CheZ
modulation are sufficient to produce the desired gain.
Although the model analyzed herein is specific to signal
transduction in bacterial chemotaxis, the structure of the
network in Fig. 2 is very similar to those of other signal
transduction processes, such as those modeled in refs. 27–29.
This suggests that the type of analysis done here will have
applicability to other systems. A more complete discussion of
aspects of adaptation not treated here, including an evaluation
of general models of adaptation, is given in ref. 10.
This work was supported in part by National Institutes of Health
Grant GM29123 to H.G.O. and National Institutes of Health Grant
GM19559 to J.S.P.
1. Berg, H. C. (1990) Cold Spring Harbor Symp. Quant. Biol. 55,
539–545.
2. Block, S. M., Segall, J. E. & Berg, H. C. (1982) Cell 31, 215–226.
3. Stock, J. B. (1994) in Regulation of Cellular Signal Transduction
Pathways by Desensitization and Amplification, eds. Sibley, D. R.
& Houslay, M. D. (Wiley, New York), pp. 3–24.
4. Stock, J. B. & Surette, M. G. (1996) Escherichia coli and Salmo-
nella: Cellular and Molecular Biology (Am. Soc. Microbiol.,
Washington, DC).
5. Bourret, R. B., Borkovich, K. A. & Simon, M. I. (1991) Annu.
Rev. Biochem. 60, 401–441.
6. Engström, P. & Hazelbauer, G. L. (1980) Cell 20, 165–171.
7. Springer, M. S., Zanolari, B. & Pierzchala, P. A. (1982) J. Biol.
Chem. 257, 6861–6866.
8. Boyd, A. & Simon, M. I. (1980) J. Bacteriol. 143, 809–815.
9. Bray, D., Bourret, R. B. & Simon, M. I. (1993) Mol. Biol. Cell 4,
469–482.
10. Spiro, P. A. (1997) Ph.D. dissertation (Univ. of Utah, Salt Lake
City).
11. Lupas, A. & Stock, J. B. (1989) J. Biol. Chem. 264, 17337–17342.
12. Simms, S. A., Stock, A. M. & Stock, J. B. (1987) J. Biol. Chem.
262, 8537–8543.
13. Terwilliger, T. C., Wang, J. Y. & Koshland, D. E. (1986) J. Biol.
Chem. 261, 10814–10820.
14. Bray, D. & Bourret, R. B. (1995) Mol. Biol. Cell 6, 1367–1380.
15. Borkovich, K. A., Alex, L. A. & Simon, M. I. (1992) Proc. Natl.
Acad. Sci. USA 89, 6756–6760.
16. Block, S. M., Segall, J. E. & Berg, H. C. (1983) J. Bacteriol. 154,
312–323.
17. Khan, S., Castellano, F., Spudich, J., McCray, J., Goody, R., Reid,
G. & Trentham, D. (1993) Biophys. J. 65, 2368–2382.
18. Segall, J. E., Block, S. M. & Berg, H. C. (1986) Proc. Natl. Acad.
Sci. USA 83, 8987–8991.
19. Macnab, R. M. (1995) in Two-Component Signal Transduction,
eds. Hoch, J. A. & Silhavy, T. J. (Am. Soc. Microbiol., Washing-
ton, DC), pp. 181–199.
20. Kuo, S. C. & Koshland, D. E. (1989) J. Bacteriol. 171, 6279–6287.
21. Ishihara, A., Segall, J. E., Block, S. M. & Berg, H. C. (1983) J.
Bacteriol. 155, 228–237.
22. Weis, R. M. & Koshland, D. E. (1990) J. Bacteriol. 172, 1099–
1105.
23. Stewart, R. C. & Dahlquist, F. W. (1987) Chem. Rev. 87, 997–
1025.
24. Amsler, C. D. & Matsumura, P. (1995) in Two-Component Signal
Transduction, eds. Hoch, J. A. & Silhavy, T. J. (Am. Soc. Micro-
biol., Washington, DC), pp. 89–103.
25. Blat, Y. & Eisenbach, M. (1996) J. Biol. Chem. 271, 1232–1236.
26. Parkinson, J. S. & Blair, D. F. (1993) Science 259, 1701–1702.
27. Katz, B. & Thesleff, S. (1957) J. Physiol. (London) 138, 63–80.
28. Knox, B. E., Devreotes, P. N., Goldbeter, A. & Segel, L. A.
(1986) Proc. Natl. Acad. Sci. USA 83, 2345–2349.
29. De Young, G. & Keizer, J. (1992) Proc. Natl. Acad. Sci. USA 89,
9895–9899.

Robustness in simple
biochemical networks
N. Barkai & S. Leibler
Departments of Physics and Molecular Biology, Princeton University, Princeton,
New Jersey 08544, USA
.........................................................................................................................
Cells use complex networks of interacting molecular components
to transfer and process information. These ‘‘computational
devices of living cells’’1 are responsible for many important
cellular processes, including cell-cycle regulation and signal
transduction. Here we address the issue of the sensitivity of the
networks to variations in their biochemical parameters. We
propose a mechanism for robust adaptation in simple signal
transduction networks. We show that this mechanism applies in
particular to bacterial chemotaxis2–7. This is demonstrated within
a quantitative model which explains, in a unified way, many
aspects of chemotaxis, including proper responses to chemical
gradients8–12. The adaptation property10,13–16 is a consequence of
the network’s connectivity and does not require the ‘fine-tuning’
of parameters. We argue that the key properties of biochemical
networks should be robust in order to ensure their proper
functioning.
Cellular biochemical networks are highly interconnected: a per-
turbation in reaction rates or molecular concentrations may affect
numerous cellular processes. The complexity of biochemical net-
works raises the question of the stability of their functioning. One
possibility is that to achieve an appropriate function, the reaction
rate constants and the enzymatic concentrations of a network need
to be chosen in a very precise manner, and any deviation from the
‘fine-tuned’ values will ruin the network’s performance. Another
Figure 1 a, The chemotaxis network. Chemotactic ligands bind to specialized
receptors (MCP) which form stable complexes (E), with the proteins CheA and
CheW. CheA is a kinase that phosphorylates the response regulator, CheY,
whose phosphorylated form (CheYp) binds to the flagellar motor and generates
tumbling. Binding of the ligand to the receptor modifies the tumbling frequency by
changing the kinase activity of CheA. The receptor can also be reversibly
methylated. Methylation enhances the kinases activity and mediates adaptation
to changes in ligand concentration. Two proteins are involved in the adaptation
process: CheR methylates the receptor, CheB demethylates it. A feedback
mechanism is achieved through the CheA-mediated phosphorylation of CheB,
which enhances its demethylation activity. b, Mechanism for robust adaptation. E
is transformed to a modified form, Em, by the enzyme R; enzyme B catalyses the
reverse modification reaction. Em is active with a probability of am(l), which
depends on the input level l. Robust adaptation is achieved when R works at
saturation and B acts only on the active form of Em. Note that the rate of reverse
modification is determined by the system’s output and does not depend directly
on the concentration of Em (vertical bar at the end of the arrow).
Nature © Macmillan Publishers Ltd 1997
NATURE | VOL 387 | 26 JUNE 1997
letters to nature
possibility is that the key properties of biochemical networks are
robust; that is, they are relatively insensitive to the precise values of
biochemical parameters. Here we explore the issue of robustness of
one of the simplest and best-known signal transduction networks: a
biochemical network responsible for bacterial chemotaxis. Bacteria
such as Escherichia coli are able to sense (temporal) gradients of
chemical ligands in their vicinity2. The movement of a swimming
bacterium is composed of a series of ‘smooth runs’, interrupted by
events of ‘tumbling’, in which a new direction for the next run is
chosen randomly. By modifying the tumbling frequency, a bac-
terium is able to direct its motion either towards attractants or away
from repellents. A well established feature of chemoxis is its property
of adaptation10,13–16: the steady-state tumbling frequency in a
homogeneous ligand environment is insensitive to the value of
ligand concentration. This property allows bacteria to maintain
their sensitivity to chemical gradients over a wide range of attractant
or repellent concentrations.
The different proteins that are involved in chemotactic response
have been characterized in great detail, and much is known about
the interactions between them (Fig. 1a). In particular, the receptors
that sense chemotactic ligands are reversibly methylated. Biochem-
ical data indicate that methylation is responsible for the adaptation
property: changes in methylation of the receptor can compensate
for the effect of ligand on tumbling frequency. Theoretical models
proposed in the past assumed that the biochemical parameters are
fine-tuned to preserve the same steady-state behaviour at different
ligand concentrations17,18. We present an alternative picture in
which adaptation is a robust property of the chemotaxis network
and does not rely on the fine-tuning of parameters.
We have analysed a simple two-state model of the chemotaxis
network closely related to the one proposed previously2,19. The two-
state model assumes that the receptor complex has two functional
states: active and inactive. The active receptor complex shows a
kinase activity: it phosphorylates the response regulator molecules,
Figure 2 Chemotactic response and adaptation. The system activity, A, of a
model system (the reference system described in Methods) which was subject to
a series of step-like changes in the attractant concentration, is plotted as a
function of time. Attractant was repeatedly added to the system and removed
after 20 min, with successive concentration steps of l of 1, 3, 5 and 7 mM. Note the
asymmetry to addition compared with removal of ligand, both in the response
magnitude and the adaptation time. The chemotactic drift velocity of this system
is presented in the inset. Inset: the different curves correspond to gradients V̄l ¼ 0,
0.01, 0.025 and 0.05 mM/mm. An average change in receptor occupancy of less
than 1% per second is sufficient to induce a mean drift velocity of the order of
microns per second.
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letters to nature
which then bind to the motors and induce tumbling. The receptor
complexes can be either in the active or in the inactive state,
although with probabilities that depend on both their methylation
level and ligand occupancy. The average complex activity can be
considered as the output of the network, whereas its input is the
concentration of the ligand. A quantitative description of the model
consists of a set of coupled differential equations describing inter-
actions between protein components (Box 1).
The two-state model correctly reproduces the main features of
bacterial chemotaxis. When a typical model system is subject to a
step-like change in attractant concentration, l (Fig. 2), it is able to
respond and to adapt to the imposed change. The adaptation is
nearly perfect for all ligand concentrations. The addition (removal)
of attractant causes a transient decrease (increase) in system activity,
and thus of tumbling frequency. We observe a strong asymmetry in
Box 1 Two-state model of the bacterial chemotactic network
The main component of the two-state model2,19 is the receptor complex,
MCP þ CheA þ CheW (Fig. 1a), considered here as a single entity, E. The
complex is assumed to have two functional states—active and inactive. A
receptor complex in the active state shows a kinase activity of CheA; by
phosphorylating the response regulators, CheY, it sends a tumbling signal to
the motors. The output of the network is thus the average number of receptors
in the active state, the system activity A. It is assumed that this quantity
determines the tumbling frequency of the bacteria. The transformation
between A and the tumbling frequency depends on the kinetics of CheY
phosphorylation and dephosphorylation, as well as on the interaction of
CheY with the motors, which are not considered explicitly in the present
model.
The receptor complexes are assumed to exist in different forms. Consider
a complex methylated on m sites (m ¼ 1; … M). Such a complex can either be
occupied or unoccupied by the ligand. We denote the concentration of these
complexes by E0m and Eum, respectively. Each form of the receptor complex can
be in the active state with a probability depending on both its methylation level
and its ligand occupancy. We assume that an occupied receptor complex has
the probability a0m of being in the active state; for an unoccupied receptor, this
probability is am. If l is the ligand concentration, B(R) the concentration of CheB
(CheR), and {EumB} the concentration of the EumCheB complex and so on, the
model reactions can then be illustrated schematically (see figure).
The differential equations describing our model can be written in a
standard way from the figure. For instance, the kinetic equation for Eum is
dEum
¼ 2 kl lEum þ k 2 l E0m þ
dt
ÿ 12 8 9 8 93
1 2 dm;0 2 ab am Eum B þ db Eum B þ kr Eum 2 1 R þ
ÿ 1h ÿ 1 8 9 8 9i
1 2 dmM 2 ar am E R þ a9r 1 2 am E R þ db E R þ kb E
u
m
u
m
u
m
u
mþ1
ÿ 1
m ¼ 1; … M
Nature © Macmillan Publishers Ltd 1997
914
the response to the addition compared with the removal of ligand.
This asymmetry has been observed experimentally14. The chemo-
tactic response of the system has been measured by the average drift
velocity in the presence of a linear gradient of attractant (Fig. 2,
inset). The system is very sensitive: an average change in the receptor
occupancy of ,1% per second is enough to induce a drift velocity of
,1 micron per second.
Figure 3a illustrates the most striking result of the model: we have
found that the system shows almost perfect adaptation for a wide
range of values of the network’s biochemical parameters. Typically,
one can change simultaneously each of the rate constants several-
fold and still obtain, on average, only a few per cent deviation from
perfect adaptation. For instance, over 80 per cent of model systems,
obtained from a perfectly adaptive one by randomly changing all of
its biochemical parameters by a factor of two, still show ,15%
The presence of am in the equation is due the fact that CheB demethylates
only the active receptors; we have also included two different association rate
constants of CheR to the active (ar) and the inactive (a9r) receptors (see below).
djk is the Kronecker’s delta (djk ¼ 1, when j ¼ k, and is zero otherwise). Similar
equations can be written to describe kinetics of {Eum B}, {Eum R}, E0m, {E0m B} and
{E0m R}, with additional parameters a0m defining the probabilities of E0m to be in
the active state. For fixed am and a0m the biochemical parameters of this
system include nine different rate constants (kl ; k 2 l ; ar ; a9r ; dr ; kr ; ab ; db ; kb )
and three enzyme concentrations (total concentrations of CheR, CheB and
receptor complexes).
The present model is by no means the only two-state model of the
chemotactic network that exhibits robust adaptation and proper chemotactic
response. Rather, it is one of the simplest variants that is consistent with the
experimental data on the response and adaptation of wild-type E. coli. The
main assumptions underlying this model are as follows.
X The input to the system is the ligand concentration; rapid binding (and
unbinding) of the ligand to the receptor induces an immediate change in the
activity of the complex. For simplicity, the binding affinity is assumed to be
independent of the receptor’s activity and its degree of methylation. This
assumption can be relaxed without affecting the main conclusions of our
model.
X The methylation and demethylation reaction occur on slower timescales.
A central assumption is that CheB can only demethylate active receptors. In
addition, the demethylation rate constants do not depend explicitly either on
ligand occupancy or on the methylation of the receptor, so that all active
receptors are demethylated at the same rate. In the variant of the model
discussed here, the phosphorylation of CheB is not considered explicitly; in
molecular terms, we assume that the phosphorylated form of CheB, CheBp,
does not move freely in the cell. Rather,. a CheBp molecule can only
demethylate the same receptor that has phosphorylated it. We note, however,
that this assumption can also be readily relaxed (N.B. et al., manuscript in
preparation). Robust adaptation is maintained as long as both CheB and
CheBp demethylate only the active receptors.
X The methylating enzyme, CheR, acts both on active and inactive
receptors. Here we assume that the association rate constant for this
reaction depends only on the activity of the receptor (a9r for inactive, ar for
active), whereas the dissociation rate constant dr and the catalytic rate
constant kr are the same for all forms of the receptor. This assumption can
again be relaxed in various ways. In particular, the accumulated biochemical
data indicate that CheR works at saturation and operates at its maximal
velocity. In this case, the conclusions of our model are not altered, even if dr, a9r
and ar depend on the ligand occupancy and on the methylation level21. M
B
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 letters to nature
deviation from perfect adaptation (Fig. 3a, lower panel). When
varied separately, most of the rate constants may be changed by
several orders of magnitude without inducing a significant devia-
tion from perfect adaptation.
In our model we have assumed Michaelis–Menten kinetics for
simplicity. However, we have found that cooperative effects in the
enzymatic reactions can be added without destroying the robustness
of adaptation. Similarly, robust adaptation is obtained for systems
with different numbers of methylation sites. Multiple methylation
sites are thus not required for robust adaptation, but possibly are for
allowing strong initial responses for a wide range of attractant and
repellent stimuli (N.B. et al., manuscript in preparation).
The adaptation itself, as measured by its precision (Fig. 3a), is
thus a robust property of the chemotactic network. This does not
mean, however, that all the properties are equally insensitive to
variations in the network parameters. For instance, Fig. 3b shows
that the adaptation time, t, which characterizes the dynamics of
relaxation to the steady-state activity, displays substantial variations
in the altered systems. Robustness is thus a characteristic of specific
network properties and not of the network as a whole: whereas some
properties are robust, others can show sensitivity to changes in the
network parameters.
Plots similar to the ones depicted in Fig. 3 can be obtained in
quantitative experiments. A large collection of chemotactic
mutants can be analysed for variations in the biochemical rate
constants of the chemotactic network components. Alternatively,
the rate constants of the enzymes could be systematically modified
or their expression varied. At the same time, their various physio-
logical characteristics can be measured, such as steady-state
tumbling frequency, precision of adaptation, adaption time, and
so on. In this way, the predictions of the model can be quantitatively
checked.
What features of the chemotactic network make the adaptation
property so robust? We propose here a general and simple mechan-
ism for robust adaptation. Let us introduce this mechanism for one
of the simplest networks (Fig. 1b), which can be viewed either as an
‘adaptation module’, or, as a simplifying reduction of a more
complex adaptive network, such as the one presented for bacterial
chemotaxis. Consider an enzyme, E, which is sensitive to an external
signal l, such as a ligand. Each enzyme molecule is at equilibrium
between two functional states: an active state, in which it catalyses a
reaction, and an inactive state, in which it does not. The signal level l
affects the equilibrium between two functional states of the enzyme:
we suppose that a change in l causes a rapid response of the system
by shifting this equilibrium. Thus, l is the input of this signal
transduction system and the concentration of active enzymes (that
is, the system activity, A) can be considered as its output. The
enzyme E can be reversibly modified, for example by addition of
methyl or phosphate groups. The modification of E affects the
probabilities of the active and inactive states, and hence can
compensate for the effect of the ligand. In general, then, Figure 3 Robustness of adaptation. a, The precision of adaptation, P, and b,
Aðl Þ ¼ aðl ÞE þ am ðl ÞEm , where Em and E are the concentrations adaptation time, t, to a step-like addition of saturating amount of attractant are
of the modified and unmodified enzyme, respectively, and am(l) plotted as a function of the total parameter variation k, for an ensemble of model
and a(l) are the probabilities that the modified and unmodified systems (see Methods). The time evolution of the system activity A is depicted in
enzyme is active. After an initial rapid response of the system to a the inset for the reference system (solid curve) and for an altered model system,
change in the input level, l, slower changes in the system activity obtained by randomly increasing or decreasing by a factor of two all biochemical
proceed according to the kinetics of enzyme modification. parameters of the reference system (dashed curve). Each point in the top graphs
The system is adaptive when its steady-state activity, Ast, is in a and b corresponds to a different altered system, out of the total number of
independent of l. A mechanism for adaptation can be readily 6,157. The reference system is denoted by a black diamond; the particular altered
obtained by assuming a fine-tuned dependence of the biochemical system from the inset is denoted by an open square. Bottom graphs: a, the
parameters on the signal level, l. This kind of mechanism has been probability that P is larger than 0.95; b, the probability that t deviates from the
proposed for an equivalent receptor system17,18. A mechanism for adaptation time of the reference system (,10 min) by less than 5% (solid curve)
robust adaptation, on the other hand, can be obtained when the and by a factor 5 (dashed curve). c, ‘Individuality’ in the chemotaxis model. The
rates of the modification and the reverse-modification reactions inverse steady-state activity A 2 1 is plotted as a function of the adaptation time, t.
depend solely on the system activity, A, and not explicitly on the Each point represents an altered system, obtained from the reference system
concentrations Em and E. This system can be viewed as a feed-back (arrow) by varying the concentration of CheR (between 100 to 300 molecules per
system, in which the output A determines the rates of modification cell).

Nature © Macmillan Publishers Ltd 1997

NATURE | VOL 387 | 26 JUNE 1997 915
letters to nature
reactions, which in turn determine the slow changes in A. With such
activity-dependent kinetics, the value of the steady-state activity,
Ast, is independent of the ligand level, therefore the system is
adaptive. Activity-dependent kinetics can be achieved in a variety
of ways. As a simple example, consider a system for which only the
modified enzyme can be active (a ¼ 0); the enzyme R, which
catalyses the modification reaction E → Em , works at saturation,
and the enzyme B, which catalyses the reverse-modification reaction
Em → E, can only bind to active enzymes. In this case, the modifica-
tion rate is constant at all times, whereas the reverse modification
rate is a simple function of the activity
dEm A activity. Mechanisms of robust adaptation similar to the one
¼ V Rmax 2 V Bmax ð1Þ
dt Kb þ A
where VBmax and VBmax are the maximal velocities of the modification
and the reverse-modification reactions, respectively, and Kb is
the Michaelis constant for the reverse modification reaction; we
have assumed V Rmax , V Bmax . For simplicity, we have assumed
that the enzymes follow Michaelis–Menten (quasi-steady-state)
kinetics. The functioning of the feedback can now be analysed: the
system activity is continuously compared to a reference stead-state
value
V Rmax
Ast ¼ K b :
V Bmax 2 V Rmax
For A , Ast, the amount of modification increases, leading to an
increase in A; for A . Ast, the modification decreases, leading to a
decrease in A. In this way, the system always returns to its steady-
state value of activity, exhibiting adaptation. Moreover, with these
activity-dependent kinetics, the adaptation properties is insensitive
to the values of system parameters (such as enzyme concentrations),
so adaptation is robust.
Note, however, that the steady-state activity itself, which is not a
robust property of the network, depends on the enzyme concentra-
tions. Thus, the mechanism presented here still provides a way to
control the system activity on long timescales, for example by
changing the expression level of the modifying enzymes while
preserving adaptation itself on shorter timescales.
A quantitative analysis demonstrates that, on methylation time-
scales, the kinetics of the two-state model of chemotaxis can, for a
wide range of parameters, be mathematically ‘reduced’ to the simple
activity-dependent kinetics shown in equation (1) (N.B. et al.,
manuscript in preparation). Robust adaptation thus follows natu-
rally as consequence of the simple mechanism described above. The
deviations from perfect adaptation (Fig. 3) are in fact connected to
departures from the assumptions underlying this mechanism (such
as V Rmax , V Bmax ). This simple mechanism suggests that the various
detailed assumptions about the system’s biochemistry can be easily
altered, provided that the activity-dependent kinetics of receptor
modifications is preserved. All variants of the model obtained in this
way still exhibit robust adaptation (N.B. et al., manuscript in
preparation).
Two main observations argue in favour of a robust, rather than a
fine-tuned, adaptation mechanism for chemotaxis. First, the adap-
tation property is observed in a large variety of chemotactic
bacterial populations. It is easier to imagine how a robust mechan-
ism allows bacteria to tolerate genetic polymorphism, which may
change the network’s biochemical parameters. In addition, in
genetically identical bacteria some features of the chemotactic
response, such as the values of adaptation time and of steady-
state tumbling frequency, vary significantly from one bacterium to
another, while the adaptation property itself is preserved20. This
‘individuality’ can be readily explained in the framework of the
present model. The concentrations of some cellular proteins, for
example the methylating enzyme CheR, are very low2, and thus may
Nature © Macmillan Publishers Ltd 1997
916
be subject to considerable stochastic variations. In consequence,
both adaptation time and steady-state tumbling frequency, which
are not robust properties of the network, should vary significantly.
Moreover, the present model predicts that both these quantities
should show a strong correlation in their variation (Fig. 3c), which
has been observed experimentally20.
How general are the results presented here? In addition to
explaining response and adaptation in chemotaxis, the present
model accounts, in a unifying way, for other taxis behaviour of
bacteria mediated by the same network. Indeed, as the network’s
dynamics is solely determined by the system activity, the system will
respond and adapt to any environmental change that affects this
introduced above could apply to a wider class of signal transduction
networks. Robustness may be a common feature of many key
cellular properties and could be crucial for the reliable performance
of many biochemical networks. Robust properties of a network will
be preserved even if its components are modified through random
mutations, or are produced in modified quantities. Systems whose
key properties are robust could have an important advantage in
having a larger parameter space in which to evolve and to adjust to
environmental changes.
The degree of robustness in many biochemical networks can be
quantitatively investigated. This can be achieved by characterizing a
behavioural, a physical or biochemical property while varying
systematically the expression level and the rate constants of the
network’s components.
The complexity of biological systems introduce several concep-
tual and practical difficulties, however. Among the most important
is the difficulty of isolating smaller subsystems that could be
analysed separately. For instance, in the present analysis, we have
neglected the existence of different types of receptors and any
crosstalk between them. We have also disregarded the interactions
between the chemotaxis network and other components of the cell.
In addition, the complexity and stochastic variability of biological
networks may preclude their complete molecular description. Rate
constants and concentrations of many enzymes can only be mea-
sured outside their natural cellular environment and many other
network parameters remain unknown. Robustness may provide a
way out of both these quandaries: robust properties do not depend
on the exact values of the network’s biochemical parameters and
should be relatively insensitive to the influence of the other
subsystems. It should then be possible to extract some of the
principles underlying cell function without a full knowledge of
the molecular detail. M
.........................................................................................................................
Methods
Numerical integration of the kinetics equations defining the two-state model
(see Box 1) was used to investigate its properties. Computer programs in Cþþ
language were executed on an SGI (R4000) workstation using a standard
routine (lsode from LLNL). Typical CPU time for finding a numerical solution
of a model system is of the order of 1 min. A particular model system was
obtained by assigning values to the rate constants and the total enzyme
concentrations. Most of our results were obtained for a reference system
defined by the following biochemical parameters: the equilibrium binding
constant of ligand to receptor is 1 mM and the time constant for the reaction is
1 ms (k1 ¼ 1 ms 2 1 mM 2 1 , k 2 1 ¼ 1 ms 2 1 ). CheR methylates both active and
inactive receptors at the same rate, with a Michaelis constant of 1.25 mM, and a
time constant of 10 s (ar ¼ a9r ¼ 80 s 2 1 mM 2 1 , dr ¼ 100 s 2 1 , kr ¼ 0:1 2 1 ),
CheB (CheBp) demethylates only active receptors with a Michaelis constant
of 1.25 mM and a time constant of 10 s (ab ¼ 800 s 2 1 mM 2 1 , db ¼ 1;000 s 2 1 ,
kb ¼ 0:1 s 2 1 ). The number of enzyme molecules per cell are: 10,000 receptor
complexes, 2,000 CheB and 200 CheR (cell volume of 1:4 3 10 2 15 l). The
probabilities that a receptor with m ¼ 1; …4 methylated sites is in its active
state are: a1 ¼ 0:1, a2 ¼ 0:5, a3 ¼ 0:75, a4 ¼ 1 if it is unoccupied, and a01 ¼ 0,
a02 ¼ 0:1, a03 ¼ 0:5, a04 ¼ 1 if it is occupied.
NATURE | VOL 387 | 26 JUNE 1997

Response and adaptation. In a typical assay, a model system was subject to a
step-like change in attractant concentration. A system in steady-state,
characterized by the system activity Ast, was perturbed by an addition or
removal of attractant. As a result, the system activity changed abruptly and then
relaxed, with the characteristic adaptation time, t, to a new steady-state value
Ast ⋅p. Here p measures the precision of adaptation; perfect adaptation corre-
sponds to p ¼ 1 (see inset in Fig. 3a).
Robustness of adaptation. The sensitivity of adaptation precision and
adaptation time to variations in the biochemical constants defining a model
system was investigated. An ensemble of altered systems was obtained from the
reference system by random modifications of its reaction rate constants and
enzymatic concentrations, k0n. Each alternation of the reference system was
characterized by the total parameter variation, k, which is defined as:
log ðkÞ ¼ SLn¼1 j log ðkn =k0n Þ j , where kn are the biochemical parameters of the
altered system. The altered system was subject to a step-like addition of
saturating concentrations of attractant (1 mM), and both the precision of
adaptation, p, and the adaptation time, t, were measured. The assay was
repeated for various reference model systems, with different values of bio-
chemical parameters and of am, and different variants of the model. The
robustness of adaptation (Fig. 3) is independent of these choices.
Chemotactic drift velocity. The behaviour of a model system in the presence
of a linear gradient of attractant, =l, was simulated. The movement of the
system was assumed to be composed of a series of smooth runs at a constant
velocity of 20 mm s 2 1 , interrupted by tumbling events. The tumbling frequency
was taken to be a sigmoidal function of the system activity (Hill coefficient,
q ¼ 2. Different values of q lead to the same qualitative picture; the sensitivity
increases with q). The trajectories were also subject to a rotation diffusion, with
D ¼ 0:125 rad2 s 2 1 (ref. 9). Attractant concentration was increasing along the x
direction, (with l ¼ 1 mM at x ¼ 0). The chemotactic drift velocity was
estimated by measuring the average x position of a hundred identical simulated
systems as a function of time.
Received 31 December 1996; accepted 17 April 1997.
1. Bray, D. Protein molecules as computational elements in living cells. Nature 376, 307–312 (1995).
2. Stock, J. B. & Surette, M. in E. coli and S. typhimurium: Cellular and Molecular Biology (ed. Neidhardt,
F. C.) 1103–1129 (American Soceity of Microbiology, Washington DC, 1996).
3. Parkinson, J. S. Signal transduction schemes of bacteria. Cell 73, 857–871 (1993).
4. Hazelbauer, G. L., Berg, H. C. & Matsumura, P. M. Bacterial motility and signal transduction. Cell 73,
15–22 (1993).
5. Bourret, R. B., Borkovich, K. A. & Simon, M. I. Signal transduction pathways involving protein
phosphorylation in prokaryotes. Annu. Rev. Biochem. 60, 401–441 (1991).
6. Adler, J. Chemotaxis in bacteria. Annu. Rev. Biochem. 44, 341–356 (1975).
7. Bray, D., Bourret, R. B. & Simon, M. I. Computer simulation of the phosphorylation cascade
controlling bacterial chemotaxis. Mol. Biol. Cell 4, 469–482 (1993).
8. Adler, J. Chemotaxis in bacteria. Science 153, 708–716 (1996).
9. Berg, H. C. & Brown, D. A. Chemotaxis in E. coli analysed by three-dimensional tracking. Nature 239,
500–504 (1972).
10. Macnab, R. M. & Koshland, D. E. The gradient-sensing mechanism in bacterial chemotaxis. Proc. Natl
Acad. Sci. USA 69, 2509–2512 (1972).
11. Block, S. M., Segall, J. E. & Berg, H. C. Impulse responses in bacterial chemotaxis. Cell 31, 215–226
(1982).
12. Koshland, D. E. A response regulator model in a simple sensory system. Science 196, 1055 (1977).
13. Berg, H. C. & Tedesco, P. M. Transient response to chemotactic stimuli in E. coli. Proc. Natl Acad. Sci.
USA 72, 3235–3239 (1975).
14. Springer, M. S., Goy, M. F. & Adler, J. Protein methylation in behavioural control mechanism and in
signal transduction. Nature 280, 279–284 (1979).
15. Koshland, D. E., Goldbeter, A. & Stock, J. B. Amplification and adaptation in regulatory and sensory
systems. Science 217, 220–225 (1982).
16. Khan, S., Spudich, J. L., McCray, J. A. & Tentham, D. R. Chemotactic signal integration in bacteria.
Proc. Natl Acad. Sci. USA 92, 9757–9761 (1995).
17. Segel, L. A., Goldbeter, A., Devreotes, P. N. & Knox, B. E. A mechanism for exact sensory adaptation
based on receptor modification. J. Theor. Biol. 120, 151–179 (1986).
18. Hauri, D. C. & Ross, J. A model of excitation and adaptation in bacterial chemotaxis. Biophys. J. 68,
708–722 (1995).
19. Asakura, S. & Honda, H. Two-state model for bacterial chemoreceptor proteins. J. Mol. Biol. 176, 349–
367 (1984).
20. Spudich, J. L. & Koshland, D. E. Non-genetic individuality: chance in the single cell. Nature 262, 467–
471 (1976).
21. Kleene, S. J., Hobson, A. C. & Adler, J. Attractants and repellents influence methylation and
demethylation of methyl-accepting proteins in an extract of E. coli. Proc. Natl Acad. Sci. USA 76,
6309–6313 (1979).
Acknowledgements. We thank J. Stock, M. Surette, A. C. Maggs, U. Alon, L. Hartwell, M. Kirschner,
A. Levine, A. Libchaber, A. Murray and T. Surrey for discussion; A. C. Maggs for help with numerical
issues; and J. Stock, M. Surette and H. Berg for introducing us to bacterial chemotaxis and pointing out
many useful references. This work has been partially supported by grants from the NIH and the NSF. N.B.
is a Rothschild Fellow and a Dicke Fellow at Princeton University.
Correspondence and requests for materials should be addressed to S.L. (e-mail: leibler@princeton.edu).
Nature © Macmillan Publishers Ltd 1997
NATURE | VOL 387 | 26 JUNE 1997
letters to nature
A family of cytokine-inducible
inhibitors of signalling
Robyn Starr*, Tracy A. Willson*, Elizabeth M. Viney*,
Leecia J. L. Murray*, John R. Rayner†,
Brendan J. Jenkins†, Thomas J. Gonda†,
Warren S. Alexander*, Donald Metcalf*,
Nicos A. Nicola* & Douglas J. Hilton*
* The Walter and Eliza Hall Institute for Medical Research and The Cooperative
Research Center for Cellular Growth Factors, Parkville, Victoria, Australia 3052
† The Hanson Centre for Cancer Research, IMVS, Adelaide, Southern Australia,
Australia 5000
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Cytokines are secreted proteins that regulate important cellular
responses such as proliferation and differentiation1. Key events in
cytokine signal transduction are well defined: cytokines induce
receptor aggregation, leading to activation of members of the JAK
family of cytoplasmic tyrosine kinases. In turn, members of
the STAT family of transcription factors are phosphorylated,
dimerize and increase the transcription of genes with STAT
recognition sites in their promoters1–4. Less is known of how
cytokine signal transduction is switched off. We have cloned a
complementary DNA encoding a protein SOCS-1, containing an
SH2-domain, by its ability to inhibit the macrophage differen-
tiation of M1 cells in response to interleukin-6. Expression of
SOCS-1 inhibited both interleukin-6-induced receptor phos-
phorylation and STAT activation. We have also cloned two rela-
tives of SOCS-1, named SOCS-2 and SOCS-3, which together with
the previously described CIS (ref. 5) form a new family of
proteins. Transcription of all four SOCS genes is increased rapidly
in response to interleukin-6, in vitro and in vivo, suggesting they
may act in a classic negative feedback loop to regulate cytokine
signal transduction.
To identify cDNAs encoding proteins capable of suppressing
cytokine signal transduction, we used an expression cloning
approach. The strategy used the murine monocytic leukaemic M1
cell line that differentiates into mature macrophages and ceases
proliferation in response to various cytokines, including interleu-
kin-6 (IL-6), and in response to the steroid, dexamethasone6,7.
Parental M1 cells were infected with the RUFneo retrovirus, into
which a library of cDNAs from the factor-dependent haemopoietic
cell line FDC-P1 had been inserted8. Retrovirally infected M1 cells
that were unresponsive to IL-6 were selected in semi-solid agar
culture by their ability to generate compact colonies in the presence
of IL-6 and geneticin. One stable IL-6-unresponsive clone, 4A2, was
obtained after examining 104 infected cells (Fig. 1). A 1.4 kilobase
pair (kbp) cDNA insert, which we have named suppressor of
cytokine signalling-1, or SOCS-1, was recovered by polymerase
chain reaction (PCR) from the retrovirus that had integrated into
genomic DNA of 4A2 cells. The SOCS-1 PCR product was used to
Figure 1 Phenotype of IL-6 unresponsive M1 cell clone, 4A2. Colonies of parental
M1 cells (left panel) and clone 4A2 (right panel) cultured in semi-solid agar for 7
days in saline or 100 ng ml 2 1 IL-6.
917