JOURNAL OF BACTERIOLOGY, Nov. 1997, p. 6573–6580
0021-9193/97/$04.0010
Copyright © 1997, American Society for Microbiology

Vol. 179, No. 21

Thermosensing Properties of Mutant Aspartate Chemoreceptors
with Methyl-Accepting Sites Replaced Singlyor Multiply by Alanine
SO-ICHIRO NISHIYAMA, TOSHIFUMI NARA,† MICHIO HOMMA, YASUO IMAE,‡
AND IKURO KAWAGISHI*
Division of Biological Science, Graduate School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-01, Japan
Received 9 June 1997/Accepted 22 August 1997

The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modificationof its four methylation sites (Gln295, Glu302, Gln309, and Glu491). Without posttranslational deamidation,
Tar has no thermosensing ability. When Gln295 and Gln309 are deamidated to Glu, the unmethylated andheavily methylated forms function as warm and cold sensors, respectively. In this study, we carried out
alanine-scanning mutagenesis of the methylation sites. Although alanine substitutions influenced the signaling
bias and the methylation level, all of the mutants retained aspartate-sensing function. Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function. In the posttranslational modification-defective
background, some of the alanine substitutions restored thermosensing ability. Warm sensors were foundamong mutants retaining two glutamate residues, and cold sensors were found among those with one or no
glutamate residue. This result suggests that the negative charge at the methylation sites is one factor that
determines thermosensor phenotypes, although the size and shape of the side chain may also be important. The
warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes
were found. Thus, the different thermosensing phenotypes that result from covalent modification of themethylation sites may reflect distinct structural states. Broader implications for the thermosensing mechanism
are also discussed.

protein CheW (14, 34). The thermosensing mechanism isthought to involve temperature-dependent changes in the
structure of the receptor-CheW-CheA ternary complex and/orin the interactions among multiple ternary complexes. Amongthe components of the ternary complex, the receptor itself isthought to be the primary thermosensor because (i) a receptorless strain never gives a thermotactic response under any
condition tested (28) and (ii) each of the four receptors characteristically functions as either a warm sensor (Tsr, Tar, andTrg) or a cold sensor (Tar in the presence of attractants [seebelow] and Tap) (21, 27, 29).
The chemo/thermoreceptors are reversibly methylated at
specific glutamate residues located in the C-terminal cytoplasmic domain (for reviews, see references 23, 32, and 40). This
methylation is a key element of the adaptation process inchemo/thermotaxis. Receptor methylation and demethylation
are catalyzed by the methyltransferase CheR and the methylesterase CheB. CheB also has a deamidase activity that converts glutamine residues at methylation sites to glutamate residues. Tar has four methylation sites, Gln295, Glu302, Gln309,
and Glu491. Throughout this report, these individual residues
will be referred to as sites 1, 2, 3, and 4, respectively, or
collectively as QEQE. Modified sets of methylation sites will
be described in a similar fashion. Sites 1, 2, and 3 are locatedon the same face of a putative a-helix (the first methylation
helix [MH1]), and site 4 is on a second helix (MH2) (Fig. 1A).
Tar is a warm sensor in the absence of aspartate, but it isconverted to a cold sensor after adapting to aspartate or maltose (27). We have recently reported that covalent modification of the methylation sites influences the thermosensing
function (28) (Fig. 1B). The unmodified form of Tar (QEQE)
mediates a chemoresponse but not a thermoresponse. It ac-

Escherichia coli responds to small changes in temperature byaltering its swimming behavior to migrate in spatial temperature gradients (22). This phenomenon, thermotaxis, is well
suited to studies of the molecular mechanism of thermosensing. Four closely related transmembrane proteins have been
identified as thermosensors (21, 27, 29). These “thermometer”
proteins were originally identified as methyl-accepting chemotaxis proteins (MCPs) and as transducers responsible for chemotaxis (for reviews, see references 23, 32 and 40). In response
to a repellent or an attractant, the receptor activates or inactivates the cytoplasmic histidine kinase CheA, which phosphorylates itself and serves as a phosphodonor for the cytoplasmic
signaling protein CheY. Phospho-CheY interacts with the flagellar motor, which otherwise rotates counterclockwise (CCW),
to cause clockwise (CW) rotation that results in loss of propulsive power and change of swimming direction (tumbling). A
warm sensor mediates attractant and repellent responses uponincreases and decreases in temperature, respectively, and acold sensor mediates the opposite responses to the same stimuli (29).
Both in the presence and in the absence of its ligand, thereceptors exist as homodimers (25) that form stable complexes
with the CheA homodimer and two molecules of the coupling* Corresponding author. Mailing address: Department of Molecular
Biology, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-01, Japan. Phone: 81-52-789-2993. Fax: 81-52-789-3001.
E-mail: i45406a@nucc.cc.nagoya-u.ac.jp.
† Present address: Faculty of Pharmaceutical Sciences, Hokkaido
University, Kita-ku, Sapporo 060, Japan.
‡ Deceased.
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J. BACTERIOL.

FIG. 1. Effects of covalent modification at methyl-accepting residues of Tar on its thermosensing function. (A) The Tar polypeptide. Two predicted a-helical regions
containing methylation sites are indicated. MH1 and MH2, the first and second methylation helices; Q and E, methylation sites; N and C, the amino and carboxy terminiof Tar; TM1 and TM2, the first and second transmembrane segments. (B) Schematic illustration of the three distinct thermosensor forms of Tar. Tar forms ahomodimer that interacts stably with CheW and CheA. Equilibrium between the two signaling states of a null thermosensor should be unaffected by temperature. By
contrast, increases in temperature should shift that of a warm sensor toward CheA inactivating (i.e., CCW rotation of flagella; smooth swimming) and that of a cold
sensor toward CheA activating (i.e., CW rotation of flagella; tumbling). CM, cytoplasmic membrane; Q, glutamine residue; E, glutamate residue; Em, methylated
glutamate residue; Null, null thermosensor form; Warm, warm-sensor form; Cold, cold-sensor form; Asp, aspartate.

quires thermosensing ability when it is deamidated by CheB:
the unmethylated form (Tar-EEEE) functions as a warm sensor, and the heavily methylated form (represented by the fully
methylated form Tar-EmEmEmEm, Em standing for a methylated glutamate residue) functions as a cold sensor. Therefore, Tar appears to exist in three distinct states, in which it
functions as a warm, cold, or null thermosensor.
To investigate this modulation of thermosensing function
further, we have constructed and characterized a series ofmutant Tar proteins whose methylation sites are substituted
with alanine singly or multiply in all possible combinations. In
the CheB2 CheR2 background, warm sensors were foundamong mutants retaining two glutamate residues and cold sensors among those with one or no glutamate residue, suggestingthat the negative charge at the methylation sites is one factor
that determines thermosensor phenotypes, although the sizeand shape of the side chain may also be important.
MATERIALS AND METHODS
Bacterial strains. E. coli HCB339 [Dtsr-7021 D(tar-tap)5201 trg::Tn10 thr leu
his met rpsL136] (45) and CP553 [Dtrg-100 zab::Tn5 D(tar-cheB)2234 Dtsr-7028](4), which lack four chemoreceptors (except Aer), were used as plasmid hosts.
Plasmids. Plasmid pAK101 (17) carries wild-type tar. Plasmids pRA111 to
pRA126, constructed in this study from pAK101, contain mutant tar genes
encoding receptors with altered methyl-accepting sites (Table 1). The following
nucleotide substitutions were introduced by site-directed mutagenesis and verified by DNA sequencing: the codon for residue 295 (methylation site 1) of Tar
was changed from CAG (Gln) to GCG (Ala); residue 302 (site 2) was changed
from GAA (Glu) to GCA (Ala); residue 309 (site 3) was changed from CAG(Gln) to GCG (Ala); and residue 491 (site 4) was changed from GAA (Glu) to
GCA (Ala).
Site-directed mutagenesis. Site-directed mutagenesis was performed by themethod of Kunkel et al. (18). Oligonucleotides for mutagenesis and DNA sequencing were synthesized at the Center for Gene Research at Nagoya University.
Measurement of chemoresponse. Temporal stimulation assays were carriedout essentially as described previously (42). Cells were grown at 30°C withvigorous shaking in tryptone-glycerol medium (1% Bacto Tryptone [Difco Laboratories, Detroit, Mich.], 0.5% NaCl, 0.5% glycerol) supplemented with ampicillin (50 mg/ml). After 4 h of cultivation, cells were harvested by centrifugation
at room temperature and washed with motility medium consisting of 10 mM

TABLE 1. Thermoresponses mediated by the mutant receptors
without modification
Thermoresponsec

Plasmid

Methylaccepting site

Aspartate
response(M)bNo
addition

1Aspartated

pAK101
pRA111
pRA112
pRA113
pRA114
pRA121
pRA122
pRA123
pRA124
pRA125
pRA120pRA119
pRA118
pRA117
pRA116
pRA126

QEQE
AEQE
QAQE
QEAE
QEQA
AAQE
AEAE
AEQA
QAAE
QAQA
QEAA
AAAE
AAQA
AEAA
QAAA
AAAA

1026
1027
1026
1026
1025
1026
1027
1025
1026
1025
1026
1026
1025
1025
1026
1025

2
Warm
2
2
2
2
Warm
2
2
2
2
2
2
2
2
2

2
2
Cold
2
Cold
2
2
2
2
Cold
2
2
Cold
2
2
Cold

a

a
Each plasmid was introduced into strain CP553 (MCP2 CheB2 CheR2). All
cells harboring these plasmids showed a tumbly swimming pattern, as assayed atroom temperature in motility medium without any chemoeffector.
bConcentration of aspartate giving a half-maximal chemoresponse.
c2, no thermoresponse; Warm, warm-sensor response; Cold, cold-sensor response.
d
Aspartate was added at the concentrations given in the aspartate response
column.

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6575

RESULTS

FIG. 2. In vivo methylation patterns of the wild-type and mutant Tar proteinsin the presence or absence of aspartate. HCB339 (CheB1 CheR1) cells expressing wild-type or mutant Tar (indicated with their methylation sites before modification [QEQE, QEAE, etc.]) were incubated with 3.3 mM [methyl-3H]methionine at 30°C for 30 min. H2O (indicated with 2) or aspartate (final
concentration, 10 mM; indicated with 1) was then added to the samples, whichwere incubated for a further 15 min. Note that the glutamine residues at sites 1
and 3 are converted into glutamate residues by CheB amidase activity in the hoststrain.

potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 10 mM sodium DL-lactate(pH 7.0), and 0.1 mM methionine. Cells were resuspended in motility medium
and kept at room temperature. Immediately after addition of an attractant or arepellent to the cell suspension, the swimming pattern of the cells was observedwith a dark-field microscope and recorded on videotape. In an image integrated for 1 s by using an Argus-10 image processor (Hamamatsu Photonics,
K.K., Hamamatsu, Japan), smooth-swimming and tumbling cells gave line andblurred dot traces, respectively. The smooth-swimming fraction was defined as
the percent fraction of smooth-swimming cells per total swimming cells that does
not include cells attached to glass surface. The threshold concentrations ofchemicals for tactic responses were estimated from their dose-response curves.
Synthetic sodium L-aspartate, obtained from ICN Pharmaceuticals, Inc. (Plainview, N.Y.), was used for measurements of the chemoresponse.
Measurement of thermoresponse. Thermoresponses were assayed as described previously (28). A cell suspension was prepared as described above. If
necessary, an appropriate concentration of the attractant aspartate or the repellent glycerol was added just before observation. A drop of the suspension wasplaced on a glass slide mounted on a temperature control apparatus (21). The
temperature was changed from about 16°C to 27°C and returned to 16°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 above.
Analysis of methylation pattern. Methylation of Tar was measured essentiallyas described previously (42). In brief, cells in MLC medium (10 mM potassium
phosphate buffer [pH 7.0], 0.1 mM EDTA, 10 mM DL-lactate, 0.2 mg of chloramphenicol per ml) were incubated at 30°C with vigorous shaking. After 30 min,3.3 mM [methyl-3H]methionine (12 Ci/mmol; Dupont, NEN Research Products,
Boston, Mass.) was added to the cells, which were further shaken for 30 min. To
enhance the methylation of Tar, aspartate was added to a final concentration of
10 mM, and the cells were shaken for 15 min. The reaction was terminated byadding trichloroacetic acid (final concentration, 5%). Methyl-labeled proteins
were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) followed by fluorography.

Methylation levels of alanine-substituted mutant Tar. Plasmids carrying the wild-type or mutant tar genes (Table 1) wereintroduced into E. coli host strains that are defective for thefour chemoreceptor genes tsr, tar, trg, and tap. These strainsthemselves do not respond to temperature changes, although
they have the recently identified aer gene, coding for the related receptor responsible for aerotaxis (2, 33). Thus, the only
thermoreceptors present are encoded by the plasmid-borne tar
genes. To make sure that all mutant proteins were expressed,
we examined whole-cell extracts by Western blotting using
antiserum raised against Tsr-T156C (16), which cross-reacts
with Tar (data not shown). All mutant Tar receptors were
present at levels similar to that of wild-type Tar.
Plasmid pAK101 (wild-type tar) and its mutant derivatives
were introduced into a receptorless strain HCB339 (CheB1
CheR1). The resulting transformants were incubated with[methyl-3H]methionine in the presence of 10 mM aspartate,
and the whole-cell extracts were subjected to SDS-PAGE followed by fluorography (Fig. 2). It should be noted that themore a receptor is methylated, the faster it migrates in a gel (3,5, 11, 12). As shown in Fig. 2, the mutant Tar proteins gave
fewer bands, corresponding to the decrease in the number ofglutamate residues available for methylation. All of the mutant
proteins behaved similarly (data not shown). Since all of themutant receptors except Tar-AAAA were methylated in response to aspartate, the substitutions with alanine apparently
do not severely affect the chemotactic signaling functions ofTar, since methylation of the receptor is a consequence of
signal production. The methylation patterns of the four mutant
Tar proteins with single substitutions were different from eachother (Fig. 2), indicating that the methyl-accepting abilities ofthe four residues are different, as had been demonstrated previously for Tar (37–39, 44) and for Trg (30).
Swarming ability of strains expressing mutant Tar proteins.
To assess their functions as chemosensors, we examined theabilities of the mutant Tar proteins to support swarming ofcolonies in tryptone semisolid agar (Fig. 3). HCB339 cells
expressing any mutant Tar spread slower than those expressing
wild-type Tar (QEQE) but faster than those without any chemoreceptor. Colonies of cells expressing plasmid-encoded TarAEQE, -QAQE, -QEAE, -QEQA, -AEQA, -QAQA, -AAQA,
or -AEAA formed clear chemotactic rings, but the ring diameters varied significantly among transformants carrying plasmids with these different tar alleles. The other mutant Tar

FIG. 3. Swarming behavior of cells expressing each mutant Tar receptor. An aliquot (2 ml) of fresh overnight culture of HCB339 (CheB1 CheR1) cells carrying
pAK101, its derivative pRA plasmid, or the vector pBR322 was spotted on a tryptone semisolid agar plate supplemented with ampicillin (50 mg/ml). The plate wasincubated at 30°C for 12 h. To the right of the photograph, the mutant receptors are indicated with their methylation site configurations before modification.

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NISHIYAMA ET AL.

proteins seemed not to support good chemotaxis and had substitutions at site 2, site 3, or both: cells with Tar-QEAA or-QAAA spread well but formed only faint chemotactic rings,
and cells expressing any other mutant proteins neither spread
well nor formed clear chemotactic rings. In particular, all threemutant receptors with substitutions at sites 2 and 3 did notsupport chemotactic swarming, whereas cells expressing any
mutant receptor that retains intact sites 2 and 3 were invariably
chemotactic. This result is consistent with the notion that thesesites play crucial roles in adaptation, since adaptation is essential for chemotaxis and requires methylation (see Discussion
for details).
The swarm diameters did not correspond to receptor methylation levels. For example, Tar-QEQA was methylated much
more poorly than Tar-QAQE and -QEAE (Fig. 2), but itsupported better spreading. Cells expressing the nonmethylatable Tar-AAAA spread poorly but still significantly better thanthose expressing Tar-AAQE, -QAAE, or -AAAE. These results suggest that the signaling bias of the receptor is more
important for spreading than its ability to be methylated. Such
nonchemotactic swarming was found previously among strains
deleted for the genes required for chemotaxis, and signaling
bias seemed to be critical for the efficiency of such swarming(46). In this regard, site 4 seems different from the other threesites. Introduction of alanine at site 4 of Tar-AAQE, -AEAE,
and -QAAE restored spreading, and even chemotaxis for TarAEAA and -AAQA, whereas alanine at the other sites had noeffect or made chemotaxis worse.
Thermosensing properties of mutant receptors with single
substitutions at the methylation sites. To assess the effects ofalanine substitutions on thermosensing, we first examined thethermosensing abilities of Tar receptors with single substitutions. Tar-AEQE, -QAQE, -QEAE, and -QEQA were expressed in strain HCB339, and the thermotactic responses of
the resulting strains were examined (Fig. 4). Although cells
expressing Tar-QEQA showed extremely tumble-biased swimming and did not give a thermoresponse in the absence of
aspartate, they produced a cold-sensor response in the presence of 0.1 mM aspartate. The other three strains showed
warm- and cold-sensor responses in the absence and presenceof aspartate, respectively. These results suggest that replacement of any single methylation site by alanine does not prevent
Tar from sensing temperature.
Thermosensing properties of mutant receptors expressed in
the modification-defective strain. We next examined thermosensing abilities of the mutant Tar proteins expressed in strain
CP553 (CheB2 CheR2). In this host, a receptor protein isneither methylated nor deamidated. In temporal stimulation
assays, Tar-QEAE, -AAQE, -AEQA, -QAAE, -QEAA,-AAAE, -AEAA, and -QAAA did not mediate a thermoresponse either in the absence or in the presence of aspartate,
though all of them still mediated chemoresponses to aspartate(Table 1). By contrast, Tar-AEQE and -AEAE mediatedwarm-sensor responses in the absence of aspartate, whereas
Tar-QAQE, -QEQA, -QAQA, -AAQA, and -AAAA mediatedcold-sensor responses in the presence of aspartate (Table 1).
Representative thermoresponses are shown in Fig. 5. Unlike
the unmodified wild-type protein (Tar-QEQE), which does not
mediate a thermoresponse (Table 1) (28), all three mutant Tar
proteins that retain both glutamine residues (sites 1 and 3)
functioned as cold sensors. These results demonstrate thatsuitable combinations of the methylation site modifications are
required for thermosensing ability and that even subtle
changes in the pattern of modification can prevent Tar fromfunctioning as a thermosensor. It should be noted that we
found no mutant Tar protein that could mediate a warm-

J. BACTERIOL.

FIG. 4. Thermoresponses of HCB339 (CheB1 CheR1) cells expressing mutant Tar proteins with single alanine substitutions. The smooth-swimming fraction of the cells was measured in the absence (A) or presence (B) of aspartate.
Aspartate was added at the concentrations giving about half-maximal chemoresponses (10 mM for Tar-AEQE, QAQE, and QEAE; 0.1 mM for QEQA).
Arrows indicate the onset of temperature changes. Temperature (C) was monitored as described in Materials and Methods. Symbols: open circles, Tar-AEQE(pRA111); open squares, Tar-QAQE (pRA112); open triangles, Tar-QEAE(pRA113); closed circles, Tar-QEQA (pRA114). Note that the glutamine residues are converted to glutamate residues in the host strain.

sensor response under certain conditions and a cold-sensor
response under others. Furthermore, we did not find any mutant form which mediated a degenerate thermoresponse. The
mutant forms that mediated cold-sensor responses had one orno glutamate residue at the methylation sites (four of five lost
glutamate at site 4), whereas those that mediated warm-sensor
responses retained two glutamate residues.
Temperature dependences of signaling by mutant receptors
expressed in the modification-defective strain. To characterizedifferential thermosensing properties further, we examined thesteady-state swimming patterns of cells expressing mutant Tar
proteins at various temperatures (Fig. 6). The smooth-swimming fraction of CP553 cells expressing Tar-AEQE or -AEAE
was measured in the absence of any chemoeffector (Fig. 6A),

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6577

FIG. 5. Thermoresponses mediated by mutant Tar proteins expressed in the CheB2 CheR2 strain CP553. Thermoresponses were measured as described in thelegend to Fig. 4. (A) Thermoresponses mediated by mutant proteins with two methyl-accepting glutamate residues in the absence of aspartate. Symbols: open circles,
Tar-QEQE (pAK101); open squares, Tar-AEQE (pRA111); open triangles, Tar-QEAE (pRA113); closed circles, Tar-AEAE (pRA122). (B) Time course of thetemperature change. (C and D) Thermoresponses mediated by mutant proteins with one or no glutamate residue in the absence (C) or presence (D) of 10 mM aspartate.
Symbols: circles, Tar-QEQA (pRA114); squares, Tar-AAAA (pRA116); triangles, Tar-AAQE (pRA121).

and that of CP553 cells expressing Tar-QEQA or -AAAA wasmeasured in the presence of 10 mM aspartate (Fig. 6B). As the
incubation temperature increased, cells expressing Tar-AEQE
or -AEAE showed a more smooth-biased swimming (Fig. 6A),
and cells expressing Tar-QEQA or -AAAA showed a more
tumble-biased swimming (Fig. 6B). These characteristics are
consistent with the thermoresponses detected in temporal
stimulation assays with these strains (Table 1).
DISCUSSION
In this study, we constructed and characterized a series ofmutant Tar receptors whose four methyl-accepting sites are
partly or fully substituted with alanine. All of the mutant Tar
receptors mediated chemotactic responses to aspartate in thetemporal assay, suggesting that substitutions with alanine do
not per se disturb the function of Tar as a chemosensor. Still,each substitution can affect the methylation state and signaling
bias during chemotaxis, and as a result, the abilities of themutant receptors to support swarming in semisolid agar varied
substantially. Since chemotaxis requires both excitation andadaptation, impaired methylation in a mutant receptor is expected to affect chemotaxis. Cells expressing any of the threemutant proteins possessing alanine substitutions at sites 2 and3 were nonchemotactic, whereas those expressing any of thefour mutant proteins retaining intact sites 2 and 3 were chemotactic, suggesting that these sites play crucial roles in adap-

tation. This conclusion is consistent with the following published results. Methylation and demethylation rates at sites 2
and 3 are much greater than those at the other two (38, 39, 44).
Furthermore, an aspartate substitution at either site 2 or 3
decreases the overall methylation rate more than a double
mutation at sites 1 and 4 does (37, 38). However, it should be
noted that an alanine substitution at site 2 or 3 is not sufficientfor the inhibition of chemotaxis. This might be because an
alanine residue mimics a methylated glutamic acid residue(30). In contrast to these inhibitory effects of substitution atsites 2 and 3, the present study shows that an alanine substitution at site 4 can significantly increase colony spreading inagar plates, and in some cases, chemotactic swarming is enhanced as well. These effects of mutations at site 4 could be
due to its location on MH2 rather than MH1.
As observed for chemotaxis, all of the mutant Tar proteins
with a single alanine substitution retained nearly normal thermosensing properties in the CheB1 CheR1 background, demonstrating that these point substitutions also do not impair
thermosensing function. When expressed in the CheB2
CheR2 background, roughly half of the mutant proteins did
not function as thermosensors, but the others restored thermosensing function: some functioned as warm sensors, and theothers functioned as cold sensors. These warm, cold, and nullthermosensor phenotypes were discrete classes; i.e., we did not
find any intermediate phenotypes. The null thermosensor
forms did not mediate thermoresponses under any conditions

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NISHIYAMA ET AL.

FIG. 6. Temperature dependences of the steady-state swimming patterns ofCP553 (CheB2 CheR2) cells expressing mutant Tar proteins. The smoothswimming fraction of the cells was measured at various temperatures in theabsence (A) or presence (B) of 10 mM aspartate. (A) Symbols: open squares,
Tar-AEQE (pRA111); closed circles, Tar-AEAE (pRA122). (B) Symbols: circles, Tar-QEQA (pRA114); squares, Tar-AAAA (pRA126).

tested. The two warm sensors (Tar-AEQE and -AEAE) had
similar temperature dependences, as did the cold sensors (TarQEQA and -AAAA). Thus, subtle differences in the methylation sites seem to affect the thermosensing property of Tar ina quantized manner. The factors that determine thermosensor
phenotypes may include the charge, size, and shape of the side
chains of residues at the methylation sites. The charge could be
the major determinant, since both mutant warm sensors possessed two glutamates, whereas the mutant cold sensors possessed one or no glutamate residue. In particular, Tar-AAAA(Fig. 5C and 6B) mediated a cold-sensor response similar tothat of the fully methylated form. However, charge is not thewhole story because the neutral side chains of glutamine andalanine produced slightly different effects on thermosensing, as
they do on signaling (31) and on methylation (39).
Cold-sensor responses were observed only in the presence of
aspartate. An attractant stimulus might be required only toshift the prestimulus behavior toward smooth swimming. Alternatively, the binding of aspartate to Tar might impose astructural constraint on the methylated forms that is required
for the cold-sensor phenotype. In fact, Tar-AAAA functioned
as a cold sensor in the presence of aspartate (Fig. 5C), but inthe absence of aspartate (Fig. 5B), it produced a very weak andtemperature-independent smooth signal. Thus aspartate, as

J. BACTERIOL.

FIG. 7. Proposed models of thermotactic signaling. (A) Model assuming
association and dissociation of the cytoplasmic domains to create the two signaling states. The thick dotted line and double-headed arrow indicate attractive
and repulsive interactions between the cytoplasmic domains. (B) Model assuming two qualitatively different types of interaction (thick dotted and shaded lines)
corresponding to the two signaling states. These interactions might occur withinor between monomers. (C) Schematic illustration of the four methylation helices(MH1, MH19, MH2, and MH29) in the homodimer of Tar. Intra- and intersubunit interactions between these helices (gray dotted lines) are likely candidates
for signaling-state-specific ones. Connections to the other parts of the protein(designated as in Fig. 1A) were indicated by gray solid lines.

well as neutralization of the methylation sites, may be required
for the cold-sensor response.
What is the nature of the thermosensing mechanism? Thisand previous studies provide some insight. It has recently beenshown that the ligand-binding affinity of Tsr was unaffected bytemperature, suggesting that thermosensing does not involve a
global change in receptor structure but rather involves local
changes within the cytoplasmic signaling domain (16). Somemodels for the chemotactic signaling mechanism of the receptor propose that association and dissociation of the cytoplasmic domains correspond to kinase-inactivating (hence CCW
rotation of flagella) and kinase-activating (hence CW rotation)
signaling states (20, 35, 36, 47), or the other way around (9, 10,41) (Fig. 7A). Models based solely on association-dissociation
can explain thermosensing as a balance between attractive andrepulsive forces that have different temperature dependences.
For example, under certain conditions, heating could increase
the interaction between domains via the hydrophobic effect or
could weaken the interaction by melting their structures. Models based on this premise would propose that the methylation

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THERMORECEPTOR WITH MUTATED METHYLATION SITES

state (and possibly the presence or absence of aspartate) determines which type of temperature dependence is dominant.
Other models argue for conformational changes within acytoplasmic domain. Tar heterodimers with a single intact cytoplasmic domain can signal in vitro (26) and in vivo (13, 43),
suggesting that the chemotactic signaling mechanism may involve interactions between dimers, conformational changes
within a single cytoplasmic domain, or both. It remains to be
seen whether thermosensing can occur in a receptor possessing
only one cytoplasmic domain per dimer. If it is assumed that
CCW- and CW-signaling forms exhibit different types of intraor intersubunit interaction (Fig. 7B), thermosensing would require that these interactions have different temperature dependences. If the CW-specific interaction is more sensitive totemperature than the CCW-specific interaction, the CW form
would become less stable beyond a certain temperature, andhence the receptor would function as a warm sensor. The
opposite relation between the CW- and CCW-specific interactions would result in a cold-sensor phenotype.
A likely candidate for intra- or intersubunit interactions thatare signaling-state specific would be the interactions between
the methylation helices themselves (Fig. 7C), although thesehelices are not required for signal production by cytoplasmic
fragments of Tsr (1). Dimerization of Tar cytoplasmic fragments through leucine zipper motifs that are fused upstream of
MH1 still allows modulation of signaling by covalent modification of methylation sites (9, 41). In these constructs, modifications at the methylation sites could cause subtle rearrangements of the MH1-MH19, MH1-MH2, or MH1-MH29 helix
pairing. Similarly, these helix-helix interactions could be modulated by displacement of MH1 during transmembrane signaling in chemotaxis (e.g., in response to aspartate), since MH1 iscoupled to the membrane-spanning helix TM2 (Fig. 7C), which
is believed to be the mobile transmembrane signaling elementin chemotaxis (6–8, 15, 19, 24). How temperature influences
the stability of these helix-helix interactions remains to be seen.
In any case, further studies of thermosensing should shed newlight on the general mechanisms of receptor signaling andadaptation.
ACKNOWLEDGMENTS
We thank Joseph J. Falke, Michael D. Manson, and John S. Parkinson for critically reading the manuscript and Robert M. Macnab for
helpful discussion.
This work was supported in part by grants-in-aid for scientific research to S.N. and I.K. from the Ministry of Education, Science andCulture of Japan.
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