THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 277, No. 2, Issue of January 11, pp. 1593–1598, 2002
Printed in U.S.A.

Sensing of Cytoplasmic pH by Bacterial Chemoreceptors Involves
the Linker Region That Connects the Membrane-spanning and the
Signal-modulating Helices*
Received for publication, October 15, 2001, and in revised form, November 6, 2001
Published, JBC Papers in Press, November 7, 2001, DOI 10.1074/jbc.M109930200

Tohru Umemura‡§, Yumi Matsumoto‡, Kouhei Ohnishi¶储, Michio Homma‡,
and Ikuro Kawagishi‡**From the ‡Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602
and ¶Kamaishi Laboratory, Marine Biotechnology Institute, Kamaishi, Iwate 026-0001, JapanThe two major chemoreceptors of Escherichia coli,
Tsr and Tar, mediate opposite responses to the samechanges in cytoplasmic pH (pHi). We set out to identify
residues involved in pHi sensing to gain insight into thegeneral mechanisms of signaling employed by the chemoreceptors. Characterization of various chimeras of
Tsr and Tar localized the pHi-sensing region to Arg259–
His267 of Tar and Gly261–Asp269 of Tsr. This region of Tarcontains three charged residues (Arg259–Ser261, Asp263,
and His267) that have counterparts of opposite charge in
Tsr (Gly261–Glu262, Arg265, and Asp269). The replacementof all of the three charged residues in Tar or Arg259–
Ser260 alone by the corresponding residues of Tsr reversed the polarity of pHi response, whereas the replacement of Asp263 or His267 did not change the polarity
but altered the time course of pHi response. These results suggest that the electrostatic properties of a shortcytoplasmic region within the linker region that connects the second transmembrane helix to the first methylation helix is critical for switching the signaling stateof the chemoreceptors during pH sensing. Similar conformational changes of this region in response to external ligands may be critical components of transmembrane signaling.

Many biological processes, such as enzyme reactions andinteractions between proteins, are influenced by pH. Therefore,
cells have to sense and adapt to changes in extracellular andintracellular pH. Despite the accumulated knowledge about
pH-dependent regulation in a wide variety of organisms, themolecular mechanisms of pH sensing are still poorly
understood.
Behavioral responses of Escherichia coli and Salmonella typhimurium to changes in pH provide a convenient system forstudying the pH-sensing mechanism. These bacteria show repellent responses to weak acids and attractant responses to
weak bases (1, 2). These responses are generated by decreases* This work was supported in part by grants-in-aid for scientificresearch from the Japan Society for the Promotion of Science (to T. U.
and I. K.) and from the Takeda Science Foundation (to I. K.). The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.
§ Present address: The Waksman Institute, 190 Frelinghuysen Rd.,
Piscataway, NJ 08854.
储 Present address: The Center for Gene Research, Kochi University,
Nangoku-shi, Kochi 783-8502, Japan.
** To whom correspondence should be addressed. Tel.: 81-52-7892993; Fax: 81-52-789-3001; E-mail: i45406a@cc.nagoya-u.ac.jp.
This paper is available on line at http://www.jbc.org

and increases of cytoplasmic pH (pHi).1 The changes in pHi
were documented by 31P nuclear magnetic resonance spectroscopy (3, 4). Usually, pHi in E. coli is maintained at around 7.5
over a range of extracellular pH (pHo) values from 5.0 to 9.0 (3,5). However, this strong pHi homeostasis can be disrupted by
the addition of weak acids or weak bases to the culture medium. When pHo is lower than pHi, weak acids can traverse themembrane in their protonated (uncharged) form and release
protons in the cytoplasm to decrease pHi. Similarly, when pHo
is higher than pHi, weak bases can traverse the membrane in
deprotonated (uncharged) forms and capture protons in thecytoplasm to increase pHi. These changes in pHi correlate wellwith tactic responses to weak acids and weak bases (4).
The signal transduction pathway for chemotaxis in E. coliand S. typhimurium has been extensively studied at the molecular level (for reviews, see Refs. 6 –9). These organisms havea set of related methyl-accepting chemoreceptors that includes
the serine receptor Tsr and the aspartate receptor Tar. These
receptors have a remarkable ability to sense a variety of stimuli, including chemoattractants, chemorepellents, temperature, and pH.
Tar and, presumably, the other chemoreceptors exist as a
homodimer of about 60-kDa subunits (10). The dimeric cytoplasmic domains form stable complexes with the histidine kinase CheA and the adaptor protein CheW (11, 12). Furthermore, the receptors, together with the CheA and CheW
proteins, cluster at a cell pole (13).
CheA phosphorylates itself and then serves as a phosphodonor for the response regulator CheY. The phosphorylated formof CheY interacts with the flagellar motor to promote 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 the associated CheA kinase,reducing the level of phospho-CheY and promoting smoothswimming.
In principle, any step in this signal transduction pathway
can be influenced by pH. In fact, the activity of the histidine
kinase CheA depends sharply on pH (14). However, the chemoreceptors have been considered to be the primary sensors forpHi, because the two major chemoreceptors, Tsr and Tar, have
opposite pHi-sensing properties (15). When expressed as a solechemoreceptor, Tsr mediates repellent and attractant responses to decreases and increases in pHi, respectively,
whereas under these conditions Tar mediates the responses

1
The abbreviations used are: pHi, cytoplasmic pH; pHo, extracellular
pH; MGM, motility medium containing glycerol; MH, methylation helix;
TM, transmembrane helix.

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Sensing of pH by Bacterial Chemoreceptors

with opposite polarity. These receptors also sense changes in
pHo (15). Moreover, the minor chemoreceptors Trg and Tap
mediate Tar- and Tsr-type responses, respectively, to changes
in pHi when they are expressed as the sole chemoreceptor (16).
In a wild-type E. coli cell, pHi responses mediated by Tsr
predominate, presumably because Tsr is the most abundantchemoreceptor. In Vibrio cholerae, the related TcpI receptor is
responsible for the pHo-dependent regulation of the expressionof the toxin-coregulated pilus (17). Therefore, the ability tosense pH might be an intrinsic property of this receptor family.
It is not clear why Tar and Tsr are different in their pHsensing properties. However, this difference is very useful inunderstanding the mechanisms of receptor signaling. Krikos etal. (15) showed that the cytoplasmic regions of Tar (residues
256 – 468) and Tsr (residues 258 – 470) are responsible for theirdifferential pHi-sensing properties. Furthermore, Oosawa et al.
(18) showed that cells expressing a C-terminal cytoplasmic
fragment of Tar (residues 256 –553) can mediate responses to a
weak acid, indicating that this fragment suffices for modulating the activity of CheA kinase in response to changes in pHi.
In this study, we constructed a new series of chimeric receptors between Tar and Tsr and identified their pHi-sensing
regions. Subsequent mutational analysis revealed the key residues: When these residues were swapped between Tar andTsr, the pHi response was inverted.
EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—All strains used in this study arederivatives of E. coli K-12. Strain HCB339 (⌬(tar-tap)5021 ⌬tsr-7028
trg::Tn10 thr leu his met rpsL136 (19)) was used as the plasmid host inchemotaxis assays. Strain DH5␣ (F⫺ ␭⫺ recA1 hsdR17 endA1 gyrA96
supE44 relA1 thi-1 ⌬(argF-lacZYA)U169 ␾80dlacZ⌬M15 (20)) was usedfor DNA manipulation.
Plasmids pFH2 and pFH5,2 which carry the tar and tsr genes, respectively, were constructed by subcloning the EcoRI fragments of
pLAN931 and pLAN1031 (21) into the EcoRI site of pSU18 (22). Plasmid pSU18 was digested with PstI and SacI, blunted with T4 DNApolymerase (Takara Shuzo), and ligated with a BglII linker to yield
plasmid pSU18⌬PS, which lacks the KpnI site in the multicloning site.2
Plasmid pFH1012 was constructed by subcloning the tar-containing
fragment of pLAN931 into the EcoRI site of pSU18⌬PS. Plasmids
pAB157, pAB160, pTsar-Cla, and pTsar-Nde carrying the chimericgenes encoding Tasr-468, Tasr-256, Tsar-468, and Tsar-256, respectively, were provided by M. I. Simon of the California Institute ofTechnology (15).3
Construction of Chimeric Chemoreceptor Genes—The pUC118-based
plasmids, which encode the Tar-Tsr hybrids Tasr-502, Tasr-441,
Tasr431, Tasr-375, and Tasr-309 (Tar residue numbering), were constructed by spontaneous homologous recombination between tar and tsr
genes placed in tandem (23). Similarly, the pUC118-based plasmids
that encode the Tsr-Tar hybrids Tsar-470, Tsar-441, Tsar-412, Tsar354, and Tsar-309 were constructed by homologous recombination between tandem tsr and tar genes. The HindIII-EcoRI fragments containing the chimeric receptor genes from these plasmids were subcloned
into the multicloning site of the medium copy number plasmid pSU18.
Oligonucleotide-based Cassette Mutagenesis of tar and tsr—To construct the sandwiched chimeric Tar (Tasar) and the point-mutant Tar
receptors, oligonucleotide-based cassette mutagenesis was carried out
as follows. We designed pairs of mutagenic oligonucleotides with overlapped regions of about 20 nucleotides at their 3⬘-ends (synthesized by
Sawadi Technology Co., Tokyo). Each pair of oligonucleotides was used
for polymerase chain reaction with ExTaq polymerase (Takara Shuzo,
Kyoto) to replace the wild-type sequence of the tar gene. The resulting
fragments were digested with NdeI and KpnI and subcloned into thecorresponding region of pFH101 to yield plasmids encoding the desired
chimeric or mutant receptors. The plasmid encoding Tsasr was constructed in a similar fashion using the NdeI and BssHII sites of the tsr
gene. The DNA sequences were verified by the dideoxy chain-termination method using reagents from Amersham Biosciences, Inc.
Swarm Assay for Chemotaxis—Swarming ability was determined as
2
3

F. Hattori, M. Homma, and I. Kawagishi, unpublished.
M. I. Simon, personal communication.

described previously (24) using Tryptone semisolid agar (1% Tryptone,
0.5% NaCl, 0.25% agar) or minimal semisolid agar (50 mM potassium
phosphate buffer (pH 7.0), 1 mM MgSO4, 1 mM glycerol, 0.1 mM each of
threonine, leucine, histidine, and methionine, 1 mg/ml thiamine, 1 mM
(NH4)2SO4, 0.25% agar) supplemented with 0.1 mM aspartic acid. Chloramphenicol (25 ␮g/ml) was added as required. Semisolid agar was
inoculated with aliquots of overnight cultures (about 4 ⫻ 106 cells) andincubated at 30 °C.
Temporal Stimulation Assay of Chemotactic Responses—The temporal stimulation assay was carried out essentially as described previously (25) with some modification. Changes in cytoplasmic pH (pHi)were elicited by the addition of sodium benzoate or acetate (2). Cells
were grown at 30 °C in TG broth (1% Tryptone, 0.5% NaCl, 0.5% (w/v)
glycerol) with chloramphenicol. Cells were harvested at late exponential phase, washed twice with motility medium (MGM) adjusted to pH
6.0 or pH 7.4 (10 mM potassium phosphate buffer, 0.1 mM EDTA, 0.1 mM
methionine, 1 mM glycerol), and resuspended in MGM at room temperature. Serine, sodium aspartate, or sodium benzoate (pH 6.0) was added
to the cell suspension, and aliquots were taken at intervals for microscopic observation. The swimming pattern of the cells was observedwith a dark-field microscope and recorded on videotape. The smoothswimming fraction of the cells was determined by analysis of the video
recording with an Argus-10 image processor (Hamamatsu Photonics
K. K., Shizuoka).
Immunoblot Analysis of Chemoreceptor Proteins—Receptor expression and methylation were examined by immunoblot analysis as described previously (26) with slight modification.
RESULTS

Characterization of the pHi-sensing Properties of Tar/Tsr
and Tsr/Tar Chimeras—To narrow down the pHi-sensing regions, we created a new series of chimeric receptors by spontaneous homologous recombination between the tar and tsr
genes placed in tandem on linearized plasmids (23). The resulting chimeric receptors, of which the N- and C-terminal regions
were derived either from Tar and Tsr, were named Tasr, using
the Tar residue at the junction point to differentiate different
hybrids. Similarly, the chimeric receptors with the N-terminal
Tsr sequences and the C-terminal Tar sequences were named
Tsar, using the same nomenclature with Tar residue numbers.4
The chimeric plasmid-borne genes were introduced into
strain HCB339, which lacks the four chemoreceptors Tsr, Tar,
Trg, and Tap. The resulting transformants were tested for theirswarming ability. In Tryptone semisolid agar, cells expressing
any Tasr or Tsar protein formed swarm rings, suggesting thatthe chimeric receptors were expressed and supported
chemotaxis.
We then examined their pHi-sensing properties in the temporal stimulation assay. Cells expressing wild-type Tar, orTasr-309, -431, -441, or -502, showed attractant responses to adecrease in pHi (Fig. 1). Cells expressing Tasr-256 showed arepellent response, like cells expressing wild-type Tsr. On theother hand, cells expressing wild-type Tsr or Tsar-309, -354,-412, or -441 showed repellent responses to a decrease in pHi.
Cells expressing Tsar-256 showed an attractant response to thesame stimulation. These results suggest that residues 256 –309
of Tar and residues 258 –311 of Tsr are responsible for pHi
sensing.
Replacement of Short Cytoplasmic Sequences of Tar by theCorresponding Tsr Sequence Inverts the Polarity of pHi Sensing—To confirm that the identified region is responsible for pHi
sensing, we constructed two “sandwich-type” chimeric receptors, Tasar-256 –278 and Tasar-256 –267, in which shortstretches of the cytoplasmic domain of Tar were replaced by thecorresponding Tsr sequences. Immunoblotting analysis demonstrated that these sandwiched chimeras were expressed at
levels comparable to wild-type Tar (Fig. 2). HCB339 cells expressing these mutant proteins were tested for their swarming
4
In this report, we adopt this system to rename the published andunpublished chimeric receptors obtained from M. I. Simon.

Sensing of pH by Bacterial Chemoreceptors

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FIG. 3. Swarming abilities of HCB339 cells expressing the
sandwiched chimeric Tar (Tasar) and the point-mutant Tar receptors. Aliquots (2 ␮l of each) of overnight cultures were spotted onto
minimal semisolid agar containing 0.1 mM aspartate and 25 ␮g/ml
chloramphenicol. The plate was incubated at 30 °C for 20 h.

FIG. 1. Chimeras between the Tsr and Tar chemoreceptors(Tsar and Tasr) and their pHi-sensing properties. Gray and white
portions indicate the amino acid sequences of Tsr and Tar, respectively.
Hatched boxes indicate transmembrane regions. The receptors marked
with asterisks were provided by M. I. Simon (15).3

FIG. 2. Expression levels and methylation patterns of the
sandwiched Tar chimeras (Tasar) and the point-mutant Tar
receptors. HCB339 cells expressing wild-type, chimeric, or mutant Tar
proteins were incubated in the presence or absence of 10 mM aspartate.
Subsequently, their cell lysates were subjected to SDS-polyacrylamide
gel electrophoresis followed by immunoblotting with anti-Tsr serum.
RG/SE/DR/HD, Tar-R259G/S260E/D263R/H267D; R259/S260E,
Tar-R259/S260E; D263R, Tar-D263R; H267D, Tar-H267D. CR, an unspecified protein cross-reacting with the serum.

ability (Fig. 3). Additionally, we tested the attractant responsesof these cells to aspartate directly by the temporal stimulation
assay (Fig. 4). In minimal semisolid agar containing aspartate,cells expressing Tasar-256 –278 produced a swarm ring comparable to that of wild-type Tar. However, Tasar-256 –267 did notsupport swarming. This defect in swarming could be due to a
defect in adaptation, because the temporal stimulation assay
showed that the chimeric receptor retained aspartate-sensing
ability.
We then examined the responses of cells expressing theTasar receptors to a decrease in pHi in the temporal stimulation assay. Typical time courses of responses mediated by wildtype Tar, wild-type Tsr, and the two Tasar chimeric receptors
are shown in Fig. 4. When the pHo is 6.0, cells expressing
wild-type Tar and Tsr showed attractant and repellent responses, respectively, after the addition of 3 mM sodium benzoate (pH 6.0). Cells expressing either of the two chimeric
receptors (Tasar-256 –278 and 256 –267) showed weak repellent responses (i.e. Tsr-type responses) (Fig. 5). Essentiallysimilar responses were observed for another weak acid, acetate(data not shown). These results indicate that the sequence from

FIG. 4. Aspartate-sensing ability of the sandwiched Tar chimeras (Tasar) and the point-mutant Tar receptors. Immediately
after the addition of 10% glycerol, various concentrations of aspartate
were added to a suspension of HCB339 cells expressing wild-type Tar(closed circles), Tasar-256 –278 (closed squares), Tasar-256 –267 (opensquares), Tar-R259G/S260E (open circles), Tar-D263R (open triangles),Tar-H267D (open diamonds), and Tar-R259G/S260E/D263R/H267D(closed triangles). After 20 s, the percentage of smooth-swimming cells
was determined. “Basal” indicates the smooth-swimming fraction in theabsence of aspartate and glycerol. For simplicity, lines are drawn only
for wild-type Tar, Tasar-256 –278, Tar-R259/S260E, and Tar-R259G/
S260E/D263R/H267D.

His256 to His267 of Tar and the corresponding sequence from
His258 to Asp269 of Tsr are involved in pHi sensing. Because thetripeptide sequence His256-Met257-Gln258 of Tar is perfectlyconserved in Tsr, the nonapeptide sequences (Arg259–His267 of
Tar and Gly261–Asp269 of Tsr), in which three residues areconserved between Tar and Tsr, must be relevant (see Fig. 7).
However, the responses mediated by the two chimeric receptors
were weaker and more transient than that mediated by wildtype Tsr. Some other residues may be required for a complete
Tsr-type response, or these chimeric receptors may be somewhat impaired in a general receptor function, such as
adaptation.
Replacement of a Short Cytoplasmic Sequences of Tsr by theCorresponding Tar Sequence also Inverts the Polarity of pHi
Sensing—To confirm that this region is responsible for the typeof pHi sensing, we also constructed the complementary sandwiched chimeric receptor, Tsasr-256 –319. Expression of thisreceptor in strain HCB339 and its function as a serine chemoreceptor were confirmed by immunoblotting and in the swarm
assay and the temporal stimulation assay, as described for the
Tasar receptors (data not shown). As expected, HCB339 cells
expressing this receptor showed attractant responses to decreases in pHi (Fig. 6). Essentially similar responses were

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Sensing of pH by Bacterial Chemoreceptors

FIG. 5. Responses of HCB339 cells expressing the sandwiched
Tar chimeras (Tasar) to a decrease in pHi. Cells expressing an
indicated receptor were suspended in MGM at pH 6.0 (closed circles) orat pH 7.4 (open circles) and incubated at room temperature for 20 min.
At the time indicated by an arrow, 3 mM sodium benzoate was added.

observed for another weak acid, acetate (data not shown).
Thus, Tsasr mediates a Tar-type (or inverted) response to adecrease in pHi.
Replacement of Two Consecutive Residues (Arg259-Ser260) of
Tar by the Corresponding Tsr Residues (Gly261-Glu262) Inverts
the Polarity of pHi Sensing—Sequence alignment of the pHisensing regions of Tar and Tsr revealed three pairs of residues
with opposite charges in Tar and Tsr (Fig. 7). These residues
seemed good candidates to be directly involved in pHi sensing.
Therefore, we replaced these three sites in Tar (Arg259/Ser260,
Asp263, and His267) by the corresponding residues in Tsr(Gly261/Glu262, Arg265, and Asp269), either individually or in
combination. Immunoblotting analysis showed that these proteins were expressed in levels comparable to wild-type Tar (Fig.
2). Their abilities to support chemotaxis were examined in theswarm assay (Fig. 3). The mutant Tar receptors supported
formation of swarm rings in minimal semisolid agar containing
aspartate.
In the temporal stimulation assay for pHi taxis, cells expressing the “triple” mutant (Tar-R259G/S260E/D263R/
H267D) or one of the “single” mutants (Tar-R259G/S260E)
showed repellent responses to a decrease in pHi, although theduration of these responses was much shorter than that mediated by wild-type Tsr (Fig. 8). Thus, swapping of the twoconsecutive residues Arg259 and Ser260 of Tar with the corresponding residues of Tsr inverted the polarity of pHi sensing.
In contrast, cells expressing Tar-D263R or Tar-H267D stillgave attractant responses to a decrease in pHi. However, theresponse mediated by the H267D mutant Tar was delayed bothin its onset and its completion than the response mediated bywild-type Tar. The response mediated by the triple mutant Tar
receptor appeared to combine the Tsr-type response of TarR259G/S260E and the slow response of Tar-H267D. Essentiallysimilar responses were observed for another weak acid, acetate(data not shown). These results suggest that residues Arg259Ser260 of Tar and Gly261-Glu262 of Tsr play important roles indetermining the polarity of pHi sensing and that His267 of Tar
and Asp269 of Tsr might also be involved in modulating the
response to changes in pHi.
DISCUSSION

In this study, we constructed and characterized various Tar/
Tsr and Tsr/Tar chimeric receptors and identified the pHisensing region of the chemoreceptors. We also identified the

FIG. 6. Responses of HCB339 cells expressing the sandwiched
Tsr chimera (Tsasr) to a decrease in pHi. Cells were suspended in
MGM at pH 6.0 (A) or at pH 7.4 (B), and responses to 3 mM (opencircles), 13.5 mM (open triangles), or 22.5 mM (open squares) sodium
benzoate were determined as described in the legend to Fig. 5. Higher
concentrations of sodium benzoate were required for clearer responses
probably because the tumble-biased signaling behavior of Tsasr.

key residue in the receptors that determines the polarity of thepHi response.
The activities of some of the cytoplasmic signal-transducing
proteins, CheA, CheB, CheR, and CheY (14, 27–29) vary withpH in vitro, and the other signal-transducing proteins may also
be affected by pH. However, because strain HCB339, whichlacks the four chemoreceptors but has all of the cytoplasmic
signal-transducing proteins, was used as the plasmid hostthroughout this study, the differences in pHi-sensing among
the strains carrying the various plasmids can be attributed tothe chemoreceptors they produce.
To define the region responsible for pH sensing, we constructed a series of chimeric receptors between Tar and Tsr,using homologous recombination between two tandem receptor
genes on linearized plasmids. This method can create a wide
variety of chimeras between two homologous proteins, because
it does not require restriction sites to be present at the chimericjunctions.
The secondary structure of the cytoplasmic region of Tar has
been predicted by the sequence alignment and its close examination of the related receptors (30, 31). The three-dimensional
structure of a cytoplasmic fragment (residues 286 –526) of Tsr
was solved (32), but the fragment does not contain the pHisensing region identified in this study (residues 258 –280 ofTsr). However, chemical modification and disulfide cross-linking of a series of mutant receptors generated by site-directed
introduction of cysteine residues (31, 33–36) have given us afairly clear picture of the three-dimensional structure of thisregion. Cysteine-scanning mutagenesis located Arg259 of Tar on
the solvent-exposed face and Ser260 on the buried face of a shorthelix connecting the second transmembrane helix (TM2) to thefirst methylation helix (MH1) (31). More recently, disulfide
scanning revealed that Ser260 of one subunit faces toward
Ser260 of the partner subunit of the Tar homodimer (35). This
arrangement might be slightly different in Tsr, because its
counterparts for Arg259 and Ser260 of Tar are Gly261 and Glu262,
respectively (Fig. 7).
Several inverted responses mediated by Tsr, Tar, and otherchemoreceptors have been reported. Responses to temperature
mediated by Tar are inverted when Tar becomes methylated
after the addition of aspartate (25, 37, 38) or when certain
mutations are introduced into TM2 (39). Responses of S. typhi-

Sensing of pH by Bacterial Chemoreceptors

1597

FIG. 7. Amino acid sequences of thepredicted pHi-sensing regions of Tar
and Tsr. These regions (256 –309) were
implicated in pHi taxis based on the results presented in Figs. 5 and 6. The second structures of these regions of Tar
have been studied by cysteine-scanningmutagenesis (31). Numbering of the residues corresponds to Tar. Shaded letters
indicate the Tsr sequence. Boxes indicate
pairs of residues that have opposite
charges in Tar and Tsr. The sandwiched
chimeras and point-mutant Tar receptors
constructed in this study are also shown.

FIG. 8. Responses of HCB339 cells expressing the point mutant
Tar receptors to a decrease in pHi. The assays were carried out asdescribed in the legend to Fig. 5.

murium to pH (2) and of E. coli to oxygen (40) are inverted
when the cheB gene is deleted. These inverted responses might
result from hypermethylation of the most abundant chemoreceptor, Tsr, in the absence of the methylesterase CheB. Tsr also
plays a role in aerotaxis (41, 42). These examples of inverted
responses seem to involve changes in the interaction between
the methylation helices (MHs) of the relevant receptors (25).
The residues identified in this study are located in the Cterminal part of the linker region, i.e. the predicted helix andturn preceding MH1, and, therefore, they may regulate thesignaling state by altering the interactions among MHs. However, it should be noted that these residues are not necessarily
pH sensor residues, although they are responsible for differential responses between Tar and Tsr.
What is the mechanism of pHi sensing? Binding of a chemoattractant to a chemoreceptor is believed to trigger a subtle
but critical inward movement of a continuous helix consistingof helix 4 of the periplasmic domain and TM2 (Ref. 43 andreferences therein), which in turn may induce a critical movement of MH, which is the central processing unit for control of
the cytoplasmic histidine kinase CheA (44). A simple scenario
may be that protonation and deprotonation at one or more
residues alter the interactions between MHs. However, thiscannot be the whole story. For example, the pKa value of theguanidino group of arginine is 12.48, which is much higher
than the physiological pH. Possible explanations to resolve thisdifficulty include: (i) The pKa of Arg256 is somehow decreased toa physiological range; (ii) Arg256 interacts with one or moreother residues that accept a proton to alter its interaction with
Arg256; and (iii) the absolute value of pHi might not be theactual signal that is sensed by the chemoreceptors. Althoughprevious studies indicated that changes in pHi serve as chemotactic stimuli (4), bacterial cells respond to other signals suchas changes in proton motive force, oxidation-reduction potential, and membrane potential (45). Therefore, changes in pHi
may affect one or more of these factors, which are sensed by thechemoreceptors. For example, changes in membrane potential
would affect the conformation of the linker region and hence
the signaling state of the chemoreceptors without involving
protonation or deprotonation of the charged residues in thelinker region. The presence of opposite charges between Tar
and Tsr would result in their opposite polarity of signaling.
The chemoreceptor forms a ternary complex with CheA andthe adaptor protein CheW (11, 12) and is localized to the pole of
the rod-shaped cell (13). The cytoplasmic fragment of Tar fused
to a leucine-zipper forms a well defined supramolecular structure in association with CheA and CheW, and the degree ofmethylation of the receptor is critical for the stability of thecomplex (46). A hexagonal receptor-kinase network has been
proposed (47) based on the crystallographic trimer of dimers of
the cytoplasmic fragment of Tsr (32). Moreover, the rate offormation of the receptor䡠CheW䡠CheA complex is greater thanthat had been expected and is affected by the ligand, raising
the possibility that assembly/disassembly of the ternary complex is involved in signaling and adaptation (48). Therefore,
interactions within and/or among receptor-kinase complexes
might also play critical roles in pH sensing, as has been suggested for receptor signaling and/or signal amplification (Ref. 9
and references therein).
In any case, the mechanism of pHi sensing seems to beclosely related to general receptor function. In this regard, it is
intriguing that the linker region is suggested to be involved insensing of pHi. Because of the high degree of sequence similarity of this regions among the related chemoreceptors and its
location between TM2 and MH1, it has been speculated that
this region may play a critical, but perhaps rather passive, rolein transmembrane signaling (e.g. Refs. 8, 9), although a model
involving two amphipathic helices in this region has been proposed (49). For the first time, this report presents experimental
evidence that associates this “linker” region to a particular
receptor function. Our results raise the possibility that changes
in pHi, and possibly other stimuli, alter the conformation ofthis part of the receptor and/or affect the way that it interacts
with other polypeptides within or outside of the receptor dimeror with the membrane. Determination, of such changes inconformation and/or interactions involved in the pHi response
in an in vitro system that reproduces the in vivo responses,
should provide a valuable insight into the general mechanismof signaling mediated by chemoreceptors.
Acknowledgments—We thank Dr. M. I. Simon for providing plasmids
and information prior to publication. We thank Dr. M. D. Manson of
Texas A&M University for critically reading the manuscript.

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