Article No. jmbi.1999.2555 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 286, 1275±1284

COMMUNICATION

Inversion of Thermosensing Property of the Bacterial
Receptor Tar by Mutations in the Second
Transmembrane Region
So-ichiro Nishiyama1, Ichiro N. Maruyama2, Michio Homma1
and Ikuro Kawagishi1*
1

Division of Biological Science
Graduate School of Science
Nagoya University, Chikusa-ku
Nagoya 464-8602, Japan2

Department of Cell Biology
The Scripps Research Institute10550 North Torrey Pines
Road, La Jolla, CA 92037, USA

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases anddecreases in temperature, respectively. However, increased levels of
methylation of the cytoplasmic domain of Tar resulting from aspartate
binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two
separate a-helices (MH1 and MH2), have suggested that intra- and/orintersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be in¯uenced by binding of aspartate,
which could trigger some displacement of MH1 through the second
transmembrane region (TM2). As an initial step toward understanding
the role of TM2 in thermosensing, we have examined the thermosensing
properties of 43 mutant Tar receptors with randomized TM2 sequences(residues 190-210). Among them, we identi®ed one mutant receptor (TarI2) that functioned as a cold sensor in the absence of aspartate. This isthe ®rst example of attractant-independent inversion of thermosensing in
Tar. Further analyses identi®ed the minimal essential divergence fromthe wild-type Tar sequence (Q191V-W192R-Q193C) required for the
inverted response. Thus, displacements of TM2 seem to in¯uence thethermosensing function of Tar.
# 1999 Academic Press*Corresponding author

Keywords: aspartate receptor; chemotaxis; methylation; temperature;
thermotaxis

Thermotaxis of Escherichia coli is one of the bestmodel systems available to study the molecularmechanisms underlying thermosensing. The relevant receptors have been identi®ed to be closely
related transmembrane receptors, which also serveas chemoreceptors (Manson, 1992; Parkinson, 1993;
Stock & Surette, 1996; Falke et al., 1997). These
receptors can be divided into the following twoclasses (Maeda & Imae, 1979; Mizuno & Imae,
1984; Nara et al., 1991). The ``warm sensors'' (Tsr,
Tar, and Trg) mediate attractant and repellent
responses upon increases and decreases of temperature, respectively, and the ``cold sensor'' (Tap)
Present address: S. Nishiyama, Biology Department
University of Utah, Salt Lake City, UT 84112, USA.
E-mail address of the corresponding author:
i45406a@nucc.cc.nagoya-u.ac.jp0022-2836/99/101275±10 $30.00/0

mediates the opposite responses to the same
stimuli.
Tar is a warm sensor in the absence of an attractant, but it is converted to a cold sensor after it has
adapted to an attractant such as aspartate (Mizuno& Imae, 1984). Covalent modi®cation of the methylation sites (Gln295, Glu302, Gln309, and Glu491;
collectively referred to as QEQE) is involved in thisinversion of thermosensing (Nara et al., 1996). The
two glutamine residues are converted to methylatable glutamate residues irreversibly by the deamidase activity of CheB. Methylation anddemethylation are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively. In a CheBÿ CheRÿ strain, the unmodi®edform of Tar (QEQE) mediates a chemoresponse but
not a thermoresponse. It acquires thermosensing
ability when it is deamidated either by CheB or bysite-directed mutagenesis: the unmethylated form
# 1999 Academic Press

1276(EEEE) functions as a warm sensor, and the fully
methylated form (EmEmEmEm; Em standing for amethylated glutamate residue) functions as a cold
sensor in the presence of aspartate. Mutational analyses of the methylation sites of Tar suggested thatmethylation of any single residue is suf®cient for
inversion of thermosensing in the presence of
aspartate (Nishiyama et al., 1999). Methylation has
also been implicated in the inverted responses
observed in other types of taxis, such as responses
to oxygen and weak acids (Taylor & Johnson,
1998). In addition to methylation, binding of aspartate to Tar may be required for its function as acold sensor (Nara et al., 1996; Nishiyama et al.,1997).
The ®rst three methylation sites are located in asingle putative a-helix (MH1), whereas the fourthsite is located in a separate helix (MH2) (Le Moual& Koshland, 1996; Danielson et al., 1997; Figure 1).
Nishiyama et al. (1997) proposed that temperaturedependent changes in intra- and/or intersubunit
interactions of MH1 and MH2 may play a critical

Transmembrane Domain of Bacterial Thermoreceptor

role in thermosensing. These interactions could bein¯uenced not only by the covalent modi®cation of
the methylation sites but also by binding of an
attractant to Tar. A bound attractant may trigger adisplacement of the second transmembrane region(TM2), which connects the periplasmic and cytoplasmic domains, and hence a displacement of
MH1. Indeed, it has been suggested that during asignaling cycle TM2 moves relative to the ®rst
transmembrane region (TM1), which pairs stably
with TM10 of the partner subunit in the Tar homodimer (Chervitz & Falke, 1995, 1996; Chervitz et al.,
1995; Lee et al., 1995; Maruyama et al., 1995;
Hughson & Hazelbauer, 1996). Such movement
may be small (Chervitz & Falke, 1996; Umemura
et al., 1998), but it may still alter interaction(s)between the cytoplasmic domains of the two subunits within a dimer. Studies using cytoplasmic
fragments of Tar (Long & Weis, 1992; Cochran &Kim, 1996; Surette & Stock, 1996; Liu et al., 1997)
and cysteine-scanned mutant Tar receptors (Chen& Koshland, 1997; Danielson et al., 1997; Le Moual

Figure 1. Schematic illustration of Tar. Tar exists as a homodimer of about 60-kDa subunit (Milligan & Koshland,1988). The dimeric cytoplasmic domains form stable complexes with the histidine kinase CheA and the adaptor protein CheW (Gegner et al., 1992; Schuster et al., 1993). CheA autophosphorylates and then serves as a phosphodonor
for the response regulator CheY. The phosphorylated form of CheY interacts with the ¯agellar motor to cause clockwise rotation, which results in tumbling behavior by the cell. Without phospho-CheY bound, the motor rotates counterclockwise, which results in smooth swimming of the cell. Binding of an attractant to a receptor inhibits theassociated CheA kinase, reducing the level of phospho-CheY and promoting smooth swimming. The hatched boxes
indicate the second transmembrane regions (TM2). The TM2 sequences of wild-type Tar (WT) and the mutant Tar-I2
receptor (I2) are aligned. Dotted lines indicate junction points between the wild-type and mutant sequences in the
hybrid receptors (see Table 2). N, amino terminus; C, carboxy terminus; MH1 and MH2, ®rst and the second methylation helices; black dots, methylation sites.

1277

Transmembrane Domain of Bacterial Thermoreceptor

Table 1. Chemo- and thermosensing properties of mutant Tar receptors with randomized TM2 sequences
Mutanta
2B
N3
N4
Y9
O14
G6
O13
O3
Q12
I3
O7
O2
O4
I2
O5
O11
R7
R8
Y40
O10
M5
Q13
R9
U421
Y5
Y37
Y62
G1
Q4
Q7
C11
M1
M3
O6
Y4
P3
G7
Q11
Y18
P4
Y34
Y49
Y50
Y53

Swimming biasb
TR
T
T
T
TR
TR
TR
TR
R
R
R
R
R
R
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
SR
S
S
S
S
S
S
S
S
S

Glyc

NT
NT
NT

NT
























ÿ
ÿ
ÿ
ÿ
ÿ




ÿ
ÿ
ÿ
ÿ
ÿ

Chemoresponsesc
Asp ( Glyc)ÿ6ÿ4

10 (10 )10ÿ5 (>10ÿ3)
10ÿ5
10ÿ5 (10ÿ5)(>10ÿ3)10ÿ6(10ÿ5)(10ÿ6)(>10ÿ3)10ÿ6 (> 10ÿ3)(10ÿ5)(10ÿ6)(10ÿ6)(10ÿ6)
10ÿ6(>10ÿ3)(>10ÿ3)(>10ÿ3)(10ÿ4)(10ÿ5)(10ÿ6)(10ÿ6)
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
(>10ÿ3)(10ÿ5)(10ÿ6)
NT
NT
NT
NT
NT
NT

ÿAsp
Warm
None
None
None
Cold
None
None?
Warm
None?
None
None
None
Cold?
Cold
None
Warm
None
None
None
None
None
None
None
None
None?
None
None
None
None
None
None
None?
None
None
None
None
None
Cold?
None
None
None
None
None
None

Thermoresponsesd

Asp
Cold
Cold
Cold
Cold
Cold?
Cold
Cold
Cold
None
Cold
None
Cold?
NT
Cold?
None
NT
NT
NT
NT
None?
None
None
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
None?
Cold
Cold
NT
NT
NT
NT
NT

a
Plasmid pTar2B and its derivatives encoding mutant Tar receptors with randomized TM2 sequences are based on the multicopy
plasmid vector pBluescriptII KS and were described previously (Maruyama et al., 1995). These plasmids were introduced into
strain HCB339 [tsr-7021 (tar-tap)5201 trg::Tn10 thr leu his met rpsL136] (Wolfe et al., 1987), which lacks the four chemo/thermoreceptors.
b
Cells were grown at 30  C with vigorous shaking in tryptone-glycerol medium (1 % (w/v) Bacto-tryptone; Difco Laboratories,
Detroit, MI, 0.5 % (w/v) NaCl, and 0.5 % (v/v) glycerol) supplemented with ampicillin (50 mg/ml). After four hours of cultivation,
cells were harvested by centrifugation at room temperature and washed with motility medium, which consists of 10 mM potassium
phosphate buffer (pH 7.0), 0.1 mM EDTA, 10 mM sodium DL-lactate (pH 7.0), and 0.1 mM methionine. Swimming patterns of cells
were assayed at room temperature without adding chemoeffector. S, smooth swimming; SR, smooth to random swimming; R, random swimming; T, tumbly swimming; TR, tumbly to random swimming.
c
Chemoresponses to glycerol (Glyc) and aspartate (Asp) were examined by temporal stimulation assays essentially as described
previously (Nishiyama et al., 1997). Cells suspended in motility medium were kept at room temperature. Immediately after adding
an attractant or a repellent to the cell suspension the swimming pattern of the cells was observed with a dark-®eld microscope andrecorded on videotape. In an image integrated for one second using an Argus-10 image processor (Hamamatsu Photonics K.K.,
Hamamatsu, Shizuoka), smooth-swimming and tumbling cells gave linear and blurred-dot traces, respectively. The smooth-swimming fraction was de®ned as the percent fraction of smooth-swimming cells per total swimming cells. Glycerol was added at 10 %.
, Responded; ÿ, did not respond; NT, not tested. Aspartate was added at various concentrations. The concentrations giving halfmaximal attractant responses are shown without parentheses when assayed in the absence of glycerol or in parentheses whenassayed in the presence of 10 % glycerol.
d
Thermoresponses were examined as described previously (Nishiyama et al., 1997). Without adding a chemoeffector (ÿAsp) or 30
minutes after 0.1 mM aspartate was added ( Asp), a drop of cell suspension in motility medium was placed on a glass slide
mounted on a temperature-control apparatus (Maeda & Imae, 1979). The temperature was changed from 16 to 27  C and returned to
16  C. Temperature changes were monitored by a constantan-chromel thermocouple inserted into the suspension. Cold, cold-sensor
response; Warm, warm-sensor response; None, no thermoresponse was observed. Question marks indicate weak or no responses,
which were dif®cult to judge.

1278

Figure 2. (a) Thermoresponses of cells expressing
wild-type Tar (*) or Tar-I2 (&) in the absence of chemoattractant. To remove additional mutations from thetar genes of pTarI2 and pTarO14, the 0.3 kb EcoRV-KpnI
fragments of the relevant derivatives of pTar2B were
subcloned into the corresponding region of pNI101, thepBR322-based plasmid carrying the wild-type tar gene,which will be described elsewhere. The smooth-swimming fraction of the cells (upper panel) was measured
as the temperature was increased and then decreased asshown in the lower panel (at one minute, 16 to 27  C; at
3.5 min, 27 to 16  C). Arrows indicate the onset of thetemperature ramps. (b) Methylation patterns of wildtype Tar and Tar-I2. The wild-type or mutant receptors
were expressed in the CheBÿ CheRÿ strain CP553 [trg100 zab::Tn5 ((tar-cheB)2234 tsr-7028] (Burrows et al.,1989) (lanes CP) or in the CheB CheR strain HCB339(lanes ÿ and ) and detected by immunoblotting analysis essentially as described previously (Okumura et al.,1998). Cells were preincubated in the absence of attractant (lanes CP and ÿ) or with 10 mM aspartate (lanes) at room temperature for 20 minutes. The cells werecollected by centrifugation and resuspended in loading

Transmembrane Domain of Bacterial Thermoreceptor

et al., 1998; Trammell & Falke, 1999) suggest thataltered interactions between the cytoplasmic
domains are associated with altered signalingstates. Changes of interactions within a single cytoplasmic domain or between dimers within a receptor cluster (Maddock & Shapiro, 1993) may also be
involved (Milligan & Koshland, 1991; Gardina &Manson, 1996; Tatsuno et al., 1996). In any case,attractant-induced movement of TM2 might
impose some physical constraint on the receptor
that in¯uences the thermosensing property of Tar.
Here, we screened for anomalous thermosensing
phenotypes among a series of mutant Tar receptors
with randomized TM2 sequences (Maruyama et al.,1995). We employed this approach because selection of mutants with altered thermotactic phenotypes is generally very dif®cult. A total of 43
mutant Tar receptors as well as the parental receptor (2B) were expressed in the receptorless strain
HCB339, and their chemo- and thermosensing
properties were examined (Table 1). In temporal
stimulation assays of chemotaxis, cells expressing
2B or 29 mutant receptors showed attractant
responses to aspartate and/or repellent responses
to glycerol. Cells expressing the remaining 14
mutant receptors did not show any chemoresponse
to glycerol or aspartate. In temporal stimulation
assays of thermotaxis, two strains (2B, O3) produced essentially normal warm-sensor responses
in the absence of attractant and cold-sensor
responses in the presence of aspartate. Six morestrains did not give warm-sensor responses in theabsence of aspartate but still exhibited cold-sensor
responses in the presence of aspartate. Otherstrains showed inverted thermoresponses. Cells
expressing the I2 or O14 mutant proteins gavecold-sensor responses in the absence of aspartate,
whereas cells expressing Q11 or O4 gave similar,
but weaker, responses. These mutants provide the®rst examples of Tar receptors that serve as cold
sensors in the absence of attractants. In the presence of aspartate, cells expressing I2 still showeda cold-sensor response, albeit a weaker one.
More than half of the mutant receptors showedaltered sensitivities to glycerol: 14 mutant receptors
mediated no or little response to 10 % glycerol, andbuffer (67 mM Tris-HCl (pH 6.8), 8 % glycerol, 1 %sodium dodecyl sulfate (SDS), 0.003 % bromophenol
blue) supplemented with 7.7 % 2-mercaptoethanol.
Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblottingwith anti-Tsr serum (Iwama et al., 1997), which crossreacts with Tar. It should be noted that multiple methylation by the CheR methyltransferase causes stepwise
increases in the mobility of a chemoreceptor during
SDS-PAGE (Boyd & Simon, 1980; Chelsky & Dahlquist,
1980; DeFranco et al., 1980; EngstroÈm & Hazelbauer,
1980). Among the four methylation sites, two glutamine
residues are deamidated by the CheB methylesterase/
deamidase to glutamate residues, a change which
decreases the mobility of Tar.

1279

Transmembrane Domain of Bacterial Thermoreceptor

eight mediated no or little response to aspartate in
the presence of 10 % glycerol (unlike wild-typeTar). These altered sensitivities may re¯ect the ability of glycerol to act on the transmembrane regionsof the receptors, probably by altering their packing(Oosawa & Imae, 1983).
We found additional mutations (D142G and
V433A) in I2 and O14 as well as their parental Tar.
These substitutions may have occurred during theinitial cloning of the tar gene (Maruyama et al.,1995). We removed these extra mutations byrecombination, using the medium-copy-number
plasmid (pBR322) rather than the original highcopy-number plasmid (pBluescript). Of the resulting ``cured'' receptors (named Tar-I2 and Tar-O14),
the former still showed an attractant-independentcold-sensor phenotype (Figure 2(a)), whereas thelatter no longer did. Therefore, we concluded thatthe altered TM2 sequence of I2 is responsible forconverting Tar from a warm sensor to a cold sensor in an attractant-independent fashion.
The attractant-independent inversion of thermosensing associated with Tar-I2 might be correlatedwith abnormally enhanced methylation. We examined the methylation pattern of Tar-I2 by immunoblotting (Figure 2(b)) and radiolabeling (notshown) analyses. In strain CP553, which lacks
CheB and CheR, Tar cannot be deamidated nor

methylated and therefore it produced a single
band in SDS-PAGE, whose mobility is presumed
to be similar to that of doubly methylated species(Figure 2(b), lanes WT CP and I2 CP). In strain
HCB339 (CheB CheR), Tar-I2 was more heavily
methylated than wild-type Tar in the absence of
aspartate (Figure 2(b) compare lanes WT ÿ andI2 ÿ). In the presence of aspartate, Tar-I2 became
still further methylated (Figure 2(b), compare lanes
I2  and I2  ÿ), demonstrating that the methylation level of Tar-I2 can still be regulated in
response to aspartate.
To examine whether enhanced methylation of
Tar-I2 is required for the attractant-independent
inversion of thermosensing, we introduced theplasmid expressing Tar-I2 into two methyltransferase-defective strains, CP553 (CheBÿ, CheRÿ) and
CP553 carrying the CheB-overproducing plasmid
pKB24 (Borkovich et al., 1992). Both strains showed
extremely smooth-biased swimming and did not
produce any thermoresponse. Moreover, neither ofthese strains responded to the repellent glycerol.
Therefore, we could not conclude that methylationof Tar-I2 is essential for the attractant-independent
inversion of thermosensing.
The TM2 sequence of Tar-I2 is extremely different from that of wild-type Tar, differing at 20 of 21
positions (Figure 1). What properties of TM2 causet-Table 2. Effects of various wild-type/I2 hybrid TM2 sequences on chemo- and thermosensing properties of
Tar

Tara

TM2b

Chemoresponsesd
Swimming
Glyc
biasc

Asp  Glyc

ThermoresponseeaThe hybrids between the wild-type and I2 tar genes were constructed using a two-step polymerase chain reaction(PCR; Landt et al., 1990). Primers used for PCR were synthesized by Sawadi Technology (Tokyo). PCR was carried out
using ExTaq polymerase (Takara Shuzo, Kyoto) with 25 cycles of denaturing at 94  C for 30 seconds, annealing at 50  C
for 1 minute, and extension at 72  C for 2 minutes. The resulting hybrid genes were subcloned into the corresponding
region of pNI101.
b
The grey and white boxes indicate the sequences of wild-type Tar and Tar-I2, respectively.
c
Swimming bias was assayed as described in Table 1.
dChemoresponses to glycerol and aspartate were assayed as described in Table 1.
eThermoresponse in the absence of aspartate was assayed as described in Table 1. Question marks indicate weak
responses.

1280

Figure 3. Thermoresponses mediated by receptors
with various hybrid TM2 regions. Thermoresponses
were measured in the absence of attractant as describedin the legend to Figure 2(a). (a) Attractant-independentcold-sensor responses mediated by Tar-I2(188-200) (*),
Tar-I2(188-196) (&), Tar-I2(197-210) (~), Tar-I2(188-196/
204-210) (*), and Tar-I2(197-203) ( & ). (b) Weak warmsensor responses mediated by Tar-I2(201-210) (*), TarI2(204-210) (&), or Tar-I2(197-200) (~).

he inversion of thermosensing? To address thisquestion, we constructed various hybrid TM2
sequences that contain different contributions fromwild-type Tar and Tar-I2. (The receptors derived
from Tar-I2 will be designated as Tar-I2(188-200)
etc. with the numbers in the parentheses indicating
residues from Tar-I2.)
All of the hybrid receptors had normal aspartate-sensing ability, but their thermosensing properties varied (Table 2). Cells expressing Tar-I2(188200) showed a cold-sensor response in the absenceof aspartate, suggesting that the NH3-terminal halfof the TM2 sequence of Tar-I2 is suf®cient for the
inversion (Figure 3(a)). Cells expressing the reciprocal hybrid, Tar-I2(201-210) gave a warm-sensor
response in the absence of aspartate, but the
response was very weak (Figure 3(b)).

Transmembrane Domain of Bacterial Thermoreceptor

Cells expressing Tar-I2(188-196) also showeda cold-sensor response without aspartate(Figure 3(a)), suggesting that the NH3-terminal
third of the TM2 sequence of Tar-I2 is suf®cient for
the inversion. Surprisingly, cells expressing thereciprocal hybrid (Tar-I2(197-210)) also produced acold-sensor response without aspartate, suggestingthat another inversion determinant exists within
the COOH-terminal two thirds of the TM2
sequence. This determinant must be within the
middle third of the TM2 sequence, because cells
expressing Tar-I2(197-203) exhibited a cold-sensor
response without aspartate, whereas cells expressing Tar-I2(204-210) showed a weak warm-sensor
response.
Tar-I2(197-203) contains the middle third of TM2
from Tar-I2. Since Tar-I2(201-210) is a weak warm
sensor in the absence of aspartate, the last half ofTM2 from Tar-I2 is not suf®cient for the inverted
response. Cells expressing Tar-I2(197-200), which
has the ®rst half of the mutant sequence of TarI2(197-203), did not give a cold-sensor response,
but rather showed a weak warm-sensor responsein the absence of aspartate (Figure 3(b)). These
results suggest that the seven residues in TarI2(197-203) that cause inversion contain elementsfrom both the ®rst and second halves of TM2.
Thus, two distinct regions of TM2 in Tar-I2 leadto attractant-independent inversion of the thermoresponse. However, cells expressing Tar-I2(188203), which has both regions causing inversion but
the last third of wild-type TM2, showed only avery weak cold-sensor response, indicating that theentire TM2 sequence contributes to thermosensing
function.
What residues are necessary for attractant-independent inversion of thermosensing in Tar-I2(188196)? All of the Tar receptors with randomized
TM2 sequences had the substitutions R188G and
F189S, which were introduced to create a BamHI
site for cassette mutagenesis (Maruyama et al.,1995). When we changed these residues back totheir wild-type counterparts (Krikos et al., 1983),
the resulting receptor (Tar-I2(190-196)) still
behaved like Tar-I2, mediating cold-sensor
responses in the absence of aspartate (data notshown).
We then reintroduced the wild-type residues one
by one into Tar-I2(190-196) starting with the
COOH-terminal residue of the mutant sequence(Table 3). Replacement of Cys193 with the wildtype residue Gln changed the thermosensing properties dramatically, whereas replacement of the
NH3-terminal residue (Val190 to Ala) had almost
no effect. These results suggest that Cys193 isrequired for the inversion of thermosensing.
Cells expressing Tar-I2(190-192) tumbled bothupon an increase and a decrease in temperature(Figure 4(a)). Tar-I2(190-192) may be an attractantindependent, methylation-dependent cold sensor.
Methylated Tar-I2(190-192) may mediate a tumbling response upon an increase in temperature,
which should cause the receptor to becomeTable 3. Effects of various substitutions in the TM2 sequence on chemo- and thermosensing properties of Tar
Host: HCB339 (CheBÿ CheRÿ)ChemoresponsedGlyc
Glyc  Asp

Host: CP553 (CheBÿ CheRÿ)Chemoresponse(Glyc)dThermoresponsee

Tara

TM2
sequenceb

Swimming
biasc

Wild typeTar-I2AQWQLAVVVRCLRL

SR
SR




< 10ÿ6 (M)< 10ÿ6

Warm
Cold


ÿ

None
None

I2(190-195)
I2(190-193)
I2(190-192)
I2(191-193)

VVRCLRVVVRCLAVVVRQLAVAVRCLAV

TR
TR
TR
SR






< 10ÿ6< 10ÿ6< 10ÿ6< 10ÿ6

Cold
Cold
Cold??
Cold

ÿ
ÿ



None
None
Cold (5%)
Cold (5%)

A190V
Q191V
W192R
Q193C
W192RAQ193CVQWQLAVAVWQLAVAQRQLAVAQWCLAVAQRCLAV

TR
TR
T
TR
TR



ND



< 10ÿ6< 10ÿ62  10ÿ6< 10ÿ6< 10ÿ6

Warm
Warm
Warm
Cold?
Cold?

NT
NT
NT



NT
NT
NT
Cold (7%)
Cold (7%)

aThermoresponsee

The pBR322-based plasmid encoding Tar-W192R (Oosawa & Simon, 1986) was provided by K. Oosawa. The other site-directed mutant tar genes were constructed as described in Table 2.
The ®rst seven residues (190-196) of the TM2 sequences are shown. Underlined letters indicate mutant residues.
Swimming bias was assayed as described in Table 1.
dChemoresponses to glycerol and aspartate were assayed as described in Table 1. ND, not determined because cells swam too tumbly to assay their repellent response.
eThermoresponse in the absence of aspartate was assayed as described in Table 1. Warm, warm-sensor response; Cold, cold-sensor response; Cold?, weak cold-sensor response; Cold??, atypicalthermoresponse, which may be regarded as attractant-independent, methylation dependent cold-sensor response (see text for detail); None, no thermoresponse; (5 % or 7 %), observed in the presence of 5 % or 7 % glycerol.
b
c

1282

Figure 4. Thermoresponses mediated by Tar receptors
with variously substituted TM2 sequences. Thermoresponses were measured in the absence of attractant as
described in the legend to Figure 2(a). (a) The atypicalthermoresponse (i.e. tumbling both upon an increase
and a decrease in temperature) mediated by Tar-I2(190-192) (*). (b) Attractant-independent cold-sensor
responses mediated by Tar-W192R Q193C (*) or TarQ193C (&).

demethylated during adaptation. The unmethylated form of Tar-I2(190-192) may serve as a warm
sensor to mediate a tumbling response upon adecrease in temperature and to become methylated
again during adaptation. It is consistent with thisinterpretation that Tar-I2(190-192) meditates acold-sensor response in strain CP553 (CheBÿ
CheRÿ), in which the methylation site of Tar is®xed to QEQE (Table 3), which mimics a methylated state of the receptor.
In a complementary approach, we replaced
wild-type residues singly with the corresponding
residues from I2 (A190V, Q191V, W192R, and
Q193C). HCB339 cells expressing Tar-A190V,
Q191V, or W192R showed warm-sensor responses
without aspartate. In contrast, cells expressing
Tar-Q193C or the double mutant W192R Q193C

Transmembrane Domain of Bacterial Thermoreceptor

gave weak cold-sensor responses in the absence of
aspartate (Figure 4(b)). Based on these results, weconclude that the tripeptide sequence Val191Arg192-Cys193 in Tar-I2(188-196) is suf®cient for
the full manifestation of the inverted thermoresponse.
Among these substitutions, Q193C might be themost critical one, since Tar with the Q193C substitution alone mediated a weak cold-sensor response
without aspartate. Gln193 is predicted to face TM1of the same subunit in the Tar homodimer(Chervitz & Falke, 1995). Therefore, the mutation
might affect the intrasubunit interaction between
TM1 and TM2. Intersubunit disul®de cross-linking
through Cys193 is unlikely to take place, becausesuch cross-linking has not been detected for TarQ193C even in the presence of an oxidant (Pakula& Simon, 1992).
Mutational analyses of the methylation sites ofTar detected three discrete types of thermoresponses among differentially methylated states ofthe protein; Tar could exist in forms that produce
warm-, cold-, or null-thermosensor responses(Nara et al., 1996; Nishiyama et al., 1997). Here wefound various intermediate thermoresponses. TarI2(201-210) and I2(202-210) appear to be weak
warm sensors, whereas I2(197-200), Tar-Q193C,
and Tar-W192R  Q193C seem to be weak cold sensors. These weak responses are reminiscent of thermoresponses in the presence of sub-saturating
concentrations of aspartate (Mizuno & Imae, 1984).
Thus, the wild-type TM2 sequence appears to be
tuned for thermosensing, although it is relatively
tolerant for mutations in terms of chemosensing(Jeffery & Koshland, 1994; Tatsuno et al., 1994;
Maruyama et al., 1995; Baumgartner & Hazelbauer,
1996). Further investigation of the role of TM2 inthermosensing should also help in elucidating therole of TM2 in chemotactic signaling.

Acknowledgments
We thank Dr K. Oosawa of Nagoya University forproviding a plasmid, Dr M. D. Manson of Texas A & M
University for critically reading the manuscript, and Dr
J. S. Parkinson of University of Utah for helpful discussion. This work was supported in part by grants-in-aid
for scienti®c research from the Ministry of Education,
Science, Sports and Culture of Japan (S.N. and I.K.) andfrom Takeda Science Foundation (I.K.).

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Edited by I. B. Holland(Received 5 October 1998; received in revised form 6 January 1999; accepted 11 January 1999)