THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 271, No. 30, Issue of July 26, pp. 17932–17936, 1996
Printed in U.S.A.

Modulation of the Thermosensing Profile of the Escherichia coli
Aspartate Receptor Tar by Covalent Modification of Its
Methyl-accepting Sites*(Received for publication, May 31, 1995, and in revised form, April 30, 1996)

Toshifumi Nara‡, Ikuro Kawagishi§, So-ichiro Nishiyama, Michio Homma, and Yasuo Imae†
From the Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, JapanThe Escherichia coli aspartate receptor Tar is involved in the thermotactic response. We have studied
how its thermosensing function is affected by the modification of the four methyl-accepting residues (Gln295,
Glu302, Gln309, and Glu491), which play essential roles in
adaptation. We found that the primary translational
product of tar mediates a chemoresponse, but not a thermoresponse, and that Tar comes to function as a thermoreceptor, once Gln295 or Gln309 is deamidated. This is
the first identification of a thermosensing-specific mutant form, suggesting that the methylation sites of Tarconstitute at least a part of the region required for thermoreception, signaling, or both. We have also investigated the inverted thermoresponse mediated by Tar in
the presence of aspartate. We found that, whereas thedeamidated-and-unmethylated form functions as awarm receptor, eliciting a smooth-swimming signal
upon increase of temperature, the heavily methylated
form functions as a cold receptor, eliciting a smoothswimming signal upon decrease of temperature. Thus, it
is suggested that Tar exists in at least three distinct
states, each of which allows it to function as a warm,
cold, or null thermoreceptor, depending on the modification patterns of its methylation sites.

Temperature is one of the crucial environmental parametersthat restrict growth and other biological activities in any organism. Therefore, virtually all organisms show some kind of
response to an increase or decrease in temperature. It is, however, generally very difficult to investigate how an organism
senses temperature shifts, since most higher organisms haveno specialized thermosensing organ, and temperature affects awide variety of cell functions. Thermotaxis in Escherichia coli,
however, has been well characterized in terms of physiology,
genetics, and biochemistry (1). Thermal stimuli are sensed by
the four closely related transmembrane receptors (2– 6).
These receptors, also known as methyl-accepting chemotaxis* This work was supported in part by grants-in-aid for scientificresearch (to I. K. and S. N.) from the Ministry of Education, Science andCulture of Japan. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
† Professor Yasuo Imae, who was a pioneering researcher in the fields
such as bacterial thermotaxis and sodium-driven flagellar motor, died
unexpectedly on July 2, 1993. This article is dedicated to him by the restof the authors with deep sorrow, respect, and affection.
‡ Present address: Faculty of Pharmaceutical Sciences, Hokkaido
University, Kita-ku, Sapporo 060, Japan.
§ To whom all correspondence should be addressed: Dept. of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya
464-01, Japan. Tel.: 81-52-789-2993; Fax: 81-52-789-3001; E-mail:i45406a@nucc.cc.nagoya-u.ac.jp.

proteins (MCPs),1 were originally identified as chemosensory
receptor/transducers (7–13). They are reversibly methylated atfour or five glutamate residues within their C-terminal cytoplasmic domains (14 –16). Methylation and demethylation are
the basis of adaptation in chemo/thermotaxis and are catalyzed
by CheR and CheB, respectively (17, 18). An attractant stimulus, whether chemical or thermal, causes elevated methylationof the receptors, which shuts off the attractant signal output. In
contrast, a repellent stimulus brings about lowered levels of
methylation to counteract the repellent signal (15). Increases
and decreases in methylation levels correspond to the magnitudes of attractant and repellent stimuli, respectively. Itshould be noted that some of the methyl-accepting glutamate
residues are derived from glutamine residues in the primarytranslational products (19, 20). CheB is also responsible for the
specific and irreversible deamidation of these groups.
Extensive genetic and biochemical studies on bacterial chemotaxis have identified all of the protein components involvedin the chemotactic signal transduction pathway from the detection of attractants and repellents to the control of swimming
behavior (for review, see Refs. 10 –13). In brief, when a chemoreceptor binds to a repellent, it activates the cytoplasmic autophosphorylating protein kinase CheA that can transfer phosphate group to the cytoplasmic signaling protein CheY and thereceptor methylesterase CheB. Phospho-CheY interacts withswitch component(s) of the flagellar motor to cause its clockwise rotation; phospho-CheB demethylates chemoreceptor molecules to shut off the tumbling signal. On the other hand,binding of an attractant to a receptor causes inhibition of CheA
kinase, which results in a decrease in CheY and CheB phosphorylation. The same signaling pathway (from the receptor to
the flagellar motor) is utilized in thermotaxis, but the mechanism underlying thermosensing has not been elucidated.
Each receptor forms a ternary complex with CheA and thecoupling protein CheW (21). Since the complex is stable in vitro(21, 22) and in vivo (23), receptor-mediated control of CheA
activity is thought to occur through structural changes within
this receptor-CheW-CheA complex. Therefore, it is reasonable
to assume that the thermosensing mechanism involves similar
structural changes in the ternary complex to those caused byincreases and decreases in temperature. It should be stressedthat temperature dependence of autophosphorylation, phosphotransfer, or dephosphorylation cannot simply explain thermotaxis. Rather, the receptors seem to be primary thermosensors (2– 6): they fall into two classes, namely warm receptors(Tsr, Tar, and Trg) and cold receptors (Tar in the presence of
attractants (see below) and Tap). A warm receptor produces
attractant and repellent signals upon increases and

1
The abbreviation used is: MCP, methyl-accepting chemotaxis
protein.

17932

Modulation of Bacterial Thermosensor

17933

decreases in temperature, respectively, whereas a cold receptor
mediates the opposite responses to the same stimuli (6).
Interestingly, two lines of evidence suggest that covalentmodification of the methyl-accepting sites might influence thethermosensing properties of the receptors. First, cheB-defective
strains, which can respond to but cannot adapt to chemotactic
stimuli, show no thermoresponse, whereas cheR-defective
strains, which cannot adapt either, retain thermosensing abilities (2, 24). The absence of a thermoresponse in cheB mutants
might be due to the absence of deamidation and/or demethylation of the receptors. Alternatively, CheB might be involved in
the thermosensing/signaling process: e.g. it might constitute athermosensor complex with the receptor, CheW and CheA.
Second, Tar produces inverted thermotactic signals in the presence of any of its specific attractants, such as aspartate andmaltose (3, 5). Whereas it usually functions as a warm receptor,
Tar appears to be converted into a cold receptor after adapting
to an attractant. Enhanced methylation of Tar has been proposed to cause this attractant-dependent conversion of thermoreceptor function.
In this study, we have studied the roles of the methyl-accepting residues (Gln295, Glu302, Gln309, and Glu491: the set of thefour residues will be referred to as [QEQE] throughout thispaper) in the thermosensing function of Tar, by examining
thermosensing profiles in the various genetic background.
EXPERIMENTAL PROCEDURES

Bacterial Strains—All of the bacterial strains used in this study arederivatives of E. coli K-12. Strains HCB339 (Dtsr-7021 D(tar-tap)5201
trg::Tn10 thr leu his met rpsL136) (25), CP553 (Dtrg-100 zab::Tn5 D(tarcheB)2234 Dtsr-7028) (26), and RP1245 (cheR)2 were provided by H. C.
Berg of Harvard University, C. Park of Korea Advanced Institute of
Science and Technology, and J. S. Parkinson of University of Utah,
respectively.
Plasmids—A pBR322-based plasmid pAK101 (27), which carries tar
and cheW, was provided by M. I. Simon of California Institute ofTechnology. Plasmids pRA130, pRA131, and pRA132, coding for the
mutant Tar proteins whose Gln295 and Gln309, Gln295 alone, and Gln309
alone, respectively, are replaced by Glu, were constructed from pAK101
in this study as follows. The codons for residues 295 and 309 werechanged from CAG (Gln) to GAG (Glu) using site-directed mutagenesis.
The mutations were verified by nucleotide sequencing. Plasmid pNI130,
which was constructed from pRA101 and pLAN931 (28), is the pBR322based plasmid that carries the same mutant tar gene as pRA130 but
lacks most of the cheW sequence.
A cheR-carrying plasmid pRAR1 was constructed as follows. The 2.3
kb NruI-PvuII fragment of plasmid pDV2,3 provided by P. Matsumura
of University of Illinois, Chicago, was blunt-ended and ligated with
EcoRV-digested pACYC184. The ligation mixture was introduced into
the cheR mutant RP1245, and chloramphenicol-resistant CheR1 transformants were selected. Plasmid DNA was extracted from the clones
and examined by restriction enzyme digestion.
DNA Manipulations—Routine DNA manipulations were carried out
according to standard procedures (29). Restriction endonucleases andother enzymes for DNA manipulations were purchased from Takara
Shuzo Co., Ltd. (Kyoto, Japan). Site-directed mutagenesis was performed by the method of Kunkel et al. (30). Oligonucleotides for mutagenesis and DNA sequencing were synthesized at the Center for Gene
Research at Nagoya University.
Measurement of Chemoresponse—Temporal stimulation assays werecarried out as described previously (5). Cells were grown at 30 °C withvigorous shaking in tryptone-glycerol broth (1% Bacto-tryptone (Difco
Laboratories, Detroit, MI), 0.5% NaCl, and 0.5% glycerol) supplemented
with ampicillin (50 mg/ml) and, if necessary, chloramphenicol (25 mg/
ml). After 5 h of cultivation, cells were harvested by centrifugation atroom temperature and washed with motility medium (10 mM potassium
phosphate buffer (pH 7.0), 0.1 mM EDTA, 10 mM sodium DL-lactate (pH7.0), and 0.1 mM L-methionine). Cells were resuspended in motility
medium and kept at room temperature. Immediately after adding an
attractant or a repellent to the cell suspension, the swimming pattern
2
3

J. S. Parkinson, personal communication.
P. Matsumura, personal communication.

FIG. 1. Absence of thermotactic response of the CP553 (CheB2
CheR2) cells carrying pAK101 which contains the wild-type tar
gene. A, swimming behavior of the cells with (closed circles) or without(open circles) 10 mM aspartate. B, the time course of the temperature.
of the cells was observed with a dark-field microscope and recorded onvideotape. The smooth-swimming fraction was measured photographically as described previously (31). The threshold concentrations ofchemicals for tactic responses were estimated from their dose-response
curves. Synthetic sodium L-aspartate obtained from ICN Pharmaceuticals, Inc. (Plainview, NY) was used throughout the measurement ofchemoresponse.
Measurement of Thermoresponse—A cell suspension was prepared as
described in the previous section. If necessary, an appropriate concentration of aspartate or glycerol was added to cells suspended in motility
medium. A drop of the suspension was placed on a glass slide mounted
on a temperature control apparatus as described previously (2). The
temperature was changed from 20 to 30 °C and returned to 20 °C.
Temperature changes were monitored by a constantan-chromel thermocouple inserted into the suspension. Changes in the swimming pattern of the cells were measured quantitatively as described in theprevious section.
RESULTS

Inhibition of the Thermosensing Ability of Tar by the Remaining Glutamine Residues—To address the role of CheB inthermosensing, we first examined whether Tar with [QEQE]
functions as a thermoreceptor, rather than [QEmQEm] (Emstanding for a methylated glutamate residue), which is expressed in cheB-defective strains and does not mediate thermoresponse. The plasmid pAK101 carrying wild-type tar was
introduced into strain CP553, which is defective in cheB andcheR as well as all four receptor genes (in all experiments inthis study, the E. coli host strains are defective in all four
receptor genes and the only receptors derive from the plasmidborne tar genes). In this strain, the methylation/amidation
state of Tar is fixed at [QEQE]. The resultant strain pAK101/
CP553 showed extremely tumbly motility and showed no thermoresponse upon temperature shift (Fig. 1A, open circles).
Even when the smooth-swimming fraction of the cells was increased by adding aspartate, no thermoresponse was seen (Fig.
1A, closed circles). Since neither the methylated [QEmQEm]
nor the unmethylated [QEQE] form of Tar has thermosensing

17934Modulation of Bacterial Thermosensor
TABLE I
Effect of the glutamine residues in the methylation sites onthermosensing ability of Tar
Plasmida

Methyl-accepting
sites of Tar

Swimming
patternb

Thermoresponsec

pAK101
pRA131
pRA132[QEQE][EEQE][QEEE]

TumbleRandomRandom2
1
1

aEach plasmid was introduced into the host strain CP553 (CheB2
CheR2).
b
Swimming patterns of the cells were assayed in the motility medium without any chemoeffector.
cThermoresponses were measured as described under “Experimental
Procedures.”

FIG. 2. Thermoresponses mediated by the deamidated form([EEEE]) of Tar. Smooth-swimming fraction of pRA130/CP553(CheB2 CheR2) cells was measured in the presence (closed circles) or
absence (open circles) of glycerol. Arrows indicate the beginning oftemperature change.

ability, failure of demethylation of the receptors in cheB mutants cannot explain their inability to respond to temperature
shift.
We then examined whether the absence of deamidating activity in cheB mutants is responsible for this phenomenon. We
constructed a tar gene coding with two glutamine codons in the
methylation sites replaced by glutamate and expressed theresulting “genetically deamidated” receptor ([EEEE]) in thecheB cheR background (CP553). The resulting cells (pRA130/
CP553) swam extremely smoothly regardless of temperaturechange (Fig. 2), but showed a normal thermoresponse when thesmooth-swimming fraction was decreased by adding glycerol, arepellent recognized by Tar (31, 32) (Fig. 2). It should be notedthat CP553 cells which do not have any receptor swam always
smoothly and did not respond to temperature change even in
the presence of glycerol (data not shown). Moreover, CheB1
CheR1 cells expressing wild-type Tsr, Tar, Trg, or Tap show
thermoresponses in the absence of glycerol (2– 6). Therefore,
temperature-dependent changes in the intracellular concentration of acetyl phosphate, which can donate a phosphate group
to CheY (33), is not likely to be the cause of thermotaxis. The
fact that even a cheB-defective strain can respond to temperature changes indicates that CheB is not a thermosensor nor apart of a thermosensor complex. Rather, Gln295, Gln309, or bothseems to prevent thermosensing function of Tar.
To examine which glutamine residue has the inhibitory effect on thermosensing ability, Gln295 and Gln309 of Tar were
replaced by Glu singly: i.e. [EEQE] and [QEEE]. In the cheB
cheR background, both of them mediated normal thermoresponses when smooth-swimming fraction of the cells was decreased by adding glycerol (Table I). These results suggest thatthe presence of both Gln295 and Gln309 in the methyl-accepting
sites prevents Tar from thermosensing in the cheB-defective
strains. We therefore concluded that in the wild-type strain,
the primary translational product of Tar ([QEQE]) cannot function as a thermoreceptor, although it has the chemosensing
ability, and that Tar acquires thermoreceptor function, once
either of its glutamine residues is deamidated by CheB.
Methylation of Tar in the Presence of Aspartate Results in Its
Conversion from a Warm to a Cold Receptor—We now turn tothe question how Tar is converted to a cold receptor in thepresence of a specific attractant such as aspartate. We firstexamined whether the genetically deamidated mutant Tar receptor ([EEEE]) has the same thermosensing characteristics as
the wild-type Tar ([QEQE]) in the CheR1 CheB1 background.
Plasmid pNI130 coding for Tar with [EEEE] was introduced

into E. coli strain HCB339, which lacks all four chemoreceptor.
As shown in Fig. 3, the resultant cells showed an essentiallysimilar aspartate dependence of thermoresponses as the wildtype Tar. In the absence of aspartate, the cells showed attractant and repellent responses to increases and decreases in temperature, respectively. Even after adapting to 1 mM aspartate(for 30 min), the cells showed similar responses to thermal
stimuli. However, when aspartate was added at a concentration of 10 mM, the cells showed a repellent response to increased
temperature (Fig. 3). They did not seem to respond to decreased
temperature, since they swam too smoothly even after prolonged incubation with aspartate. However, when glycerol wasadded further to adjust their swimming bias toward tumbling,
they showed a clear inverted thermoresponse (data not shown).
The concentration of aspartate required for the inverted
thermoresponse of pNI130/HCB339 is 1 order of magnitude
lower than that of the tsr-defective mutants (3). However, thisshould not be due to the mutations of tar (Gln295 to Glu and
Gln309 to Glu) on pNI130, since the HCB339 cells carrying thewild-type tar-containing plasmid pAK101 also require 10 mM
aspartate for the inverted thermoresponse (5). Thus, Tar with
[EEEE] can be used to assess the role of methylation in the
inversion of thermoresponses.
It has been considered that the attractant-induced methylation of Tar might be responsible for the inverted thermoresponse (1, 5). Therefore, we next compared thermosensing abilities of the methylated and unmethylated forms of Tar as
follows. As described previously, CP553 (cheB cheR) cells carrying pBR322-based plasmid pRA130 encoding Tar with
[EEEE] showed extremely smooth-biased basal swimming pattern, but normal thermoresponse in the presence of glycerol(Fig. 2). The cells never showed an inverted thermoresponse
under the conditions tested. We now constructed the pACYC184-based plasmid pRAR1 carrying the methyltransferase
gene (cheR). The pRA130/CP553 cells were further transformed with pRAR1. In the transformant (pRA130/pRAR1/
CP553) cells, Tar is expected to be heavily methylated, since
the cells retain methyltransferase but lack methylesterase. In
fact, Borkovich et al. (34) have demonstrated that Tar is fully
methylated in the cheB-defective but cheR-overexpressing
cells. Since we did not examine experimentally whether Tar
was fully methylated in the transformant cells, we can only
state that Tar must be heavily methylated (this can be predicted from the cells’ tumbly biased swimming behavior). For
simplicity, we will hereafter describe Tar as fully methylated([EmEmEmEm]) in these cells. The cells showed inverted thermoresponses in the presence of 10 mM aspartate (Fig. 4). These
results indicate that receptor methylation causes the inverted
thermoresponse mediated by Tar in the presence of aspartate.
Temperature Dependencies of the Steady-state Signaling Biases Produced by the Heavily Methylated and the Unmethylated
Forms of Tar—To further characterize the different thermosensing profiles induced by the unmethylated and the heavily

Modulation of Bacterial Thermosensor

17935

FIG. 3. Aspartate-dependent inversion of thermoresponse mediated by the genetically deamidated mutant Tar ([EEEE]).
Thermoresponse of the pNI130/HCB339 (CheB1 CheR1) cells wasmeasured in the absence of any chemoeffector (open squares) or 30 min
after addition of 1 mM (closed triangles) or 10 mM (closed circles) aspartate. Arrows indicate the beginning of temperature change.

FIG. 5. Steady-state swimming patterns of cells having
the unmethylated ([EEEE]) or the heavily methylated([EmEmEmEm]) form of Tar at various temperatures. Smoothswimming fraction of the pNI130/CP553 (CheB2 CheR2; A) or pNI130/
pRAR1/CP553 (CheB2 CheR1; B) cells was measured without additionof any chemoeffector (open squares) or 5 min after addition of 5% (closed
triangles) or 10% (closed circles) glycerol (A) or 0.1 mM aspartate (closed
circles in B).
FIG. 4. Thermoresponses mediated by the heavily methylated
form ([EmEmEmEm]) of Tar. Smooth-swimming fraction of the
pRA130/pRAR1/CP553 (CheB2 CheR1) cells was measured in the presence (closed circles) or absence (open circles) of 10 mM aspartate. Arrows
indicate the beginning of temperature change.

methylated forms of Tar, we then examined the steady-state
swimming patterns of cells with either of these two forms of
Tar at various temperatures (Fig. 5).
The cells expressing Tar with [EEEE] (pNI130/CP553:
CheB2 CheR2) showed exclusively smooth swimming at all
temperatures tested (from 15 to 35 °C). In the presence of 10%
glycerol, however, they showed a temperature-dependent
swimming pattern: almost all cells were tumbling at 15 °C, andas the temperature increased, the smooth-swimming fraction of
the cells gradually increased and finally became almost 100%
at about 30 °C or above. This profile of the unmethylated formof Tar is consistent with its warm receptor function. Moreover,the cells swam extremely smoothly in the presence of 1 or 10 mM
aspartate at all temperatures tested (data not shown). This
indicates that in the absence of methylation, aspartate cannot
convert Tar from a warm to a cold receptor.
The cells expressing Tar with [EmEmEmEm] (pNI130/
pRAR1/CP553: CheB2 CheR1) showed exclusively tumbling
behavior at all temperatures tested (from 15 to 35 °C). In thepresence of 0.1 mM aspartate, however, they showed a temperature-dependent swimming pattern: almost all cells tumbled at
30 °C or above and as the temperature decreased, the smoothswimming fraction of the cells increased and became about 75%
at 15 °C. This profile is consistent with a cold receptor function.

Thus, we conclude that the methyl esterification of the specificglutamate residues is required for the conversion of Tar froma warm to a cold receptor in the presence of aspartate.
DISCUSSION

In this study, we have demonstrated that the primary translational product of tar (Tar with [QEQE]) does not have thermosensing ability (Fig. 1), but acquires it by the deamidation ofeither Gln295 or Gln309 (Table I). The multifunctional nature ofbacterial chemoreceptors has been exploited to isolate mutants
defective only in the response for a specific chemoeffector,
leading to the identification of the amino acid residues responsible for its recognition (for Tar and Tsr, see Refs. 5 and 35– 41).
This is the first identification of a thermosensing-specific mutant form of the receptor, since it has been reported for Tar (35,42) and Tsr (43) that covalent modifications of a receptor do notseverely affect its ligand binding affinity.
One interpretation of the results presented here is that the
methylation sites of Tar constitute at least a part of the region
required for the recognition of temperature or thermotactic
signal production. In this regard, it would be interesting to test
whether a cytoplasmic fragment of Tar can mediate thermoresponse at all, since some cytoplasmic fragments of Tar and Tsr
have been shown to retain abilities to produce chemotactic
signals (44, 45).
We have also shown that the unmethylated ([EEEE]) and theheavily methylated ([EmEmEmEm]) forms of Tar function as awarm receptor and a cold receptor, respectively. The unmethylated form of Tar mediated a normal thermoresponse in thepresence of glycerol (Fig. 2). However, under no conditions

17936Modulation of Bacterial Thermosensor

tested, did it show any characteristics of a cold receptor in thecheB cheR background. In contrast, the heavily methylated
form (i.e. the genetically deamidated Tar expressed in the cheB
background) mediated an inverted thermoresponse in the presence of aspartate (Fig. 4). Under no conditions tested did it
show any characteristics of a warm receptor. Thus, the specificmethylation, and possibly the presence of aspartate or any of
the other Tar-mediated attractants also, are required for theconversion of Tar from a warm to a cold receptor.
In summary, covalent modification of the four methyl-accepting residues of Tar modulates thermosensing properties of thereceptor. These findings reinforce the notion that thermotaxis
is resulted from a temperature-dependent shift in an equilibrium between the two signaling states (corresponding tosmooth swimming and tumbling) of the receptor itself. Further
investigation of the effects of covalent modification would provide an important clue to elucidate what kind of structuralchange of the receptor plays an essential role in thermosensing.
Acknowledgments—We thank Drs. H. C. Berg, P. Matsumura, C.
Park, J. S. Parkinson, and M. I. Simon for providing us with bacterial
strains and plasmids and N. Nishioka for drawing. We especially thankDr. R. M. Macnab of Yale University for critically reading themanuscript.
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