JOURNAL OF BACTERIOLOGY, Apr. 1998, p. 1862–1868
0021-9193/98/$04.0010
Copyright © 1998, American Society for Microbiology

Vol. 180, No. 7

Chemotactic Adaptation Is Altered by Changes in the CarboxyTerminal Sequence Conserved among the Major
Methyl-Accepting Chemoreceptors
HISASHI OKUMURA, SO-ICHIRO NISHIYAMA, AKIE SASAKI, MICHIO HOMMA,
AND IKURO KAWAGISHI*
Division of Biological Science, Graduate School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-0814, Japan
Received 7 November 1997/Accepted 29 January 1998

In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible
for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB,
respectively. Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxyterminal motif (NWET/SF) that is absent in Trg and Tap. When they are expressed as sole chemoreceptors,
Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weakadaptation. It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro. To examine thephysiological significance of this binding, we characterized mutant receptors in which this pentapeptide
sequence was altered. C-terminally-mutated Tar and Tcp expressed in a receptorless E. coli strain mediatedresponses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired. Their
expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the
methylation levels of the mutant receptors increased only slightly upon addition of attractants. When CheR was
overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable tothose of wild-type Tar. Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type
Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor. These results suggest thatthe pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.
Escherichia coli and Salmonella typhimurium migrate toward
or away from certain chemicals by controlling the direction offlagellar rotation (for reviews, see references 3 and 28). Thischemotactic behavior consists of two essential aspects, excitation and adaptation. Without adaptation, cells cannot detect
temporal changes, and hence spatial gradients of concentrations of chemoeffectors, even though they can respond to theirabsolute concentrations.
Most chemoeffectors are detected by a small family ofclosely related chemoreceptors localized in the cytoplasmic
membrane (for reviews, see reference 48 and the reviews cited
above), including Tsr (for serine), Tar (for aspartate), Trg (forribose and galactose), Tap (for dipeptide), and Tcp (for citrate). Tsr, Tar, and Trg are present in both species, and Tap
and Tcp are specific to E. coli and S. typhimurium, respectively.
Tar (and presumably any other chemoreceptor as well) is a
homodimeric protein (30) comprising subunits about 60 kDa in
size, each consisting of an N-terminal periplasmic ligand-binding domain, a C-terminal cytoplasmic signaling domain, andtwo membrane-spanning segments.
Methylation and demethylation of these chemoreceptors are
the key processes of adaptation (for reviews, see reference 44
and the reviews cited above). The cytoplasmic domain of a
receptor monomer contains four to six methylatable glutamic
acid residues located in two separate regions that are predicted
to form antiparallel a-helical coiled coils (23, 26, 35, 50). The
consensus sequence for the methylation sites is Glu-Glu-X-XAla-Ser/Thr (18, 35, 38, 50). This sequence is likely to berecognized by the active site of the methyltransferase CheR
that catalyzes transfer of a methyl group from S-adenosylmethionine to the side chain of the glutamic acid residue indicated by underlining above (45). The methyl ester bond of amethylated glutamate residue is hydrolyzed by the methylesterase CheB (47). Some of the methylation sites are encoded as
glutamine residues, which CheB converts posttranslationally tomethylatable glutamic acid residues (19).
Although there is no evidence for any regulation of CheR
activity, the activity of CheB is stimulated when the protein is
phosphorylated by the histidine kinase CheA (25, 46). The
CheA homodimer forms a ternary complex with the receptor
homodimer and two molecules of the adapter protein CheW
(14, 40). Binding of a repellent or an attractant to the receptor
activates or inactivates autophosphorylation of CheA andthereby increases or decreases phosphotransfer from CheA to
its substrate proteins, CheY and CheB. Phospho-CheY induces clockwise rotation of the flagellar motor to cause the cellto tumble. When it is not bound to phospho-CheY, the motor
rotates counterclockwise to power smooth swimming. By regulating CheB activity, an attractant or a repellent stimulus
increases or decreases methylation of the receptor to attenuate
the initial counterclockwise or clockwise signal, respectively.
Such adaptation presumably results from conformationalchanges in the signaling domain caused by the methylation
state of the receptor (4, 9, 33–36, 41), although the methylation
helices are not essential for production of the chemotactic
signal itself (1). A further constraint on the role of methylationin adaptation is that covalent modification does not dramatically change the ligand-binding affinity of the receptor (4, 9, 17,
24).
Among the chemoreceptors of E. coli and S. typhimurium,
Trg and Tap are expressed at lower levels than the others (16,* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-0814, Japan. Phone: 81-52-789-2993. Fax: 81-52-789-3001.
E-mail: i45406a@nucc.cc.nagoya-u.ac.jp.
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FIG. 1. C-terminal amino acid sequences of chemoreceptors. The sequences of E. coli Tar, Tsr, Trg, and Tap and of S. typhimurium Tar (Tars) and Tcp as deduced
from the nucleotide sequences are aligned. Gaps inserted to achieve the best match are indicated by dashes. The box with the solid black border indicates the C-terminal
sequence conserved among Tar, Tsr, and Tcp, and the box with the gray border indicates the putative a-helical region (helix 10) proposed by Le Moual and Koshland(23). Methylation sites are indicated by white letters. Numbering of residues is for E. coli Tar.

42) and show weaker abilities to be methylated, to allow adaptation, and to support chemotaxis toward attractants wheneach of them is expressed as a sole chemoreceptor (12, 15, 29,
43, 51, 55). Trg and Tap are shorter and lack the C-terminal
pentapeptide sequence (NWET/SF) that is highly conserved
among Tsr, Tar, and Tcp (Fig. 1). A mutant form of S. typhimurium Tar, which has its C-terminal 35 residues removed andhence lacks the NWETF sequence, has been shown to bepoorly methylated and not to support adaptation (39), and amutant Tcp with an altered C-terminal sequence (NWESLA)
also supports little adaptation (54). Recently, Wu et al. (53)demonstrated that CheR binds to E. coli Tsr in vitro at a 1:1
molar ratio through the NWETF sequence.
To study the physiological significance of this pentapeptide
sequence, we examined the effects of mutations in this sequence on various functions of E. coli Tar and S. typhimurium
Tcp. We also examined the effects of CheR overproduction on
the truncated Tar and on wild-type E. coli Trg. The results
suggest that the C-terminal sequence, located far from themethylation sites in the primary sequence, facilitates effective
receptor methylation by recruiting CheR to a receptor patch(27) that may enable trans methylation of a receptor dimer by
CheR bound to another receptor dimer (53).
MATERIALS AND METHODS
Bacterial strains. All strains used in this work are derivatives of E. coli K-12.
Strain HCB339 [Dtsr-7021 D(tar-tap)5201 trg::Tn10 thr leu his met rpsL136] (52)lacks the four chemoreceptors, and strain CP553 [trg-100 Dtsr-7028 D(tar-cheB)leu his rpsL lac xyl ara tonA tsx thi zab::Tn5] (6) lacks CheB and CheR as well as
the four chemoreceptors.
Plasmids. A pBR322-based plasmid, pAK101 (21), carries the wild-type tar
gene of E. coli (coding for wild-type Tar, i.e., Tar[NWETF]). Its derivativeplasmid pAK101-W550Op (31), which carries a nonsense (opal) mutant tar gene(coding for Tar-W550Op, i.e., Tar[N]), was provided by K. Oosawa of Nagoya
University. Another pAK101 derivative plasmid, pNI130 (32), carries the tarQ295E,Q309E gene coding for Tar-EEEE, in which the two glutamine residues
at the methylation sites are replaced by glutamate. A pACYC184-based plasmid,
pRAR1 (32), carries the methyltransferase gene cheR and the chloramphenicolacetyltransferase gene cat (Cmr). Another pACYC184-based plasmid, pKB23(4), in which the cheR gene is placed downstream of the tac promoter, wasprovided by M. I. Simon of California Institute of Technology. A pBR322-based
plasmid, pCP31 (37), which carries the wild-type trg gene of E. coli, was provided
by G. L. Hazelbauer of Washington State University.
Plasmids carrying the wild-type and mutant tcp genes of S. typhimurium wereconstructed as follows. Plasmid pAS101 carrying the wild-type tcp gene (coding
for wild-type Tcp, i.e., Tcp[NWESF]) was constructed by subcloning the 6-kb
EcoRI fragment of plasmid pKYP29 (54) into the unique EcoRI site of thevector pSU21 (2). Plasmid pAS103 carrying the tcp-F547LA gene (coding for
Tcp-F547LA, i.e., Tcp[NWESLA]) was constructed by deleting the HindIII fragment from pAS101. Plasmid pAS103 was digested with HindIII, blunt-ended with
T4 DNA polymerase, and ligated to yield plasmid pAS104 carrying the tcpF547Am gene (coding for Tcp-F547Am, i.e., Tcp[NWES]) or ligated with the
PacI 10mer linker (dGTTAATTAAC; New England BioLabs) to yield plasmid

pAS105 carrying the tcp-F547C gene (coding for Tcp-F547C, i.e., Tcp[NWESC]).
Plasmid pOKU106 carrying the tcp-F547A gene (coding for Tcp-F547A, i.e.,
Tcp[NWESA]) was obtained by site-directed mutagenesis by the method of
Kunkel et al. (22). These mutations were verified by nucleotide sequencing.
Swarm assay. Swarm assays were performed with tryptone semisolid agar (1%tryptone, 0.5% NaCl, 0.3% agar) or minimal semisolid agar [10 mM potassium
phosphate buffer (pH 7.0), 1 mM (NH4)2SO4, 1 mM MgSO4, 1 mM glycerol, 1
mg of thiamine per ml, 0.1 mM threonine, 0.1 mM leucine, 0.1 mM histidine, 0.1
mM methionine] supplemented with 0.1 mM aspartate or 1 mM ribose. Whennecessary, 50 mg of ampicillin per ml, 25 mg of chloramphenicol per ml, and/or
1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) was added. Cell suspensions(2 ml each, about 4 3 106 cells) were spotted onto a semisolid agar plate. The
plate was then incubated at 30°C for 10 to 20 h.
Temporal stimulation assay. Temporal stimulation assays for chemotaxis werecarried out essentially as described previously (34). Cells were grown at 30°C intryptone broth (1% tryptone, 0.5% NaCl) supplemented with 0.5% (wt/vol)glycerol and, when necessary, with 50 mg of ampicillin per ml, 25 mg of chloramphenicol per ml, and/or 1 mM IPTG. Cells were harvested in late exponential
phase, washed with motility medium (10 mM potassium phosphate buffer [pH7.0], 0.1 mM EDTA, 10 mM lactate, 0.1 mM methionine), resuspended inmotility medium, and incubated at room temperature for 20 min. For doseresponse assays, the swimming patterns of the cells were observed with a darkfield optical microscope immediately after the addition of a chemoeffector. For
adaptation assays, a chemoeffector was added to the cell suspension and smallaliquots were taken at intervals for microscopic observation. The smooth-swimming fraction (SSF) of the cells was measured with an Argus-10 image processor(Hamamatsu Photonics K.K., Hamamatsu, Shizuoka, Japan).
Immunoblotting. Cells expressing a wild-type or mutant receptor were grown,
harvested, and washed as described above (see “Temporal stimulation assay”).
When necessary, chemoeffector was added to cells suspended in the motility
medium and the suspension was incubated at room temperature for 20 min. The
cells were collected by centrifugation and suspended in SDS loading buffer (67mM Tris-HCl [pH 6.8], 8% glycerol, 1% sodium dodecyl sulfate [SDS], 0.003%
bromophenol blue) supplemented with 7.7% 2-mercaptoethanol. Samples wereboiled for 3 min and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore
Japan, Tokyo) by using a semidry blotting apparatus (Biocraft, Tokyo, Japan).
Anti-Tsr-T156C serum (17) was used as the first antibody, and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (Kirkegaard & Perry
Laboratories, Gaithersburg, Md.) was used as the second antibody. The proteinantibody complexes were visualized in 5 ml of AP buffer (0.1 M Tris-HCl [pH9.5], 1 M NaCl, 5 mM MgCl2) supplemented with 33 ml of nitroblue tetrazolium
solution (50 mg of nitroblue tetrazolium per ml in 70% [vol/vol] dimethylformamide) and 16.5 ml of BCIP solution (50 mg of 5-bromo-4-chloro-3-indolylphosphate per ml in dimethylformamide).

RESULTS
Mutations in the C-terminal sequences of Tar and Tcp
cause impaired adaptation abilities. The nonsense mutant Tar
protein Tar[N] does not allow the host strain RP4372 (tsr tartap) to swarm in tryptone semisolid agar (31). The frameshift
mutant Tcp protein Tcp[NWESLA] supports little adaptationin E. coli HCB339 (tsr tar tap trg) (54). We wanted to further
characterize these mutant receptors as well as newly constructed mutant Tcp receptors (see Materials and Methods).
Plasmids carrying the mutant tar or tcp genes were introduced

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OKUMURA ET AL.

J. BACTERIOL.

FIG. 2. Swarming abilities of HCB339 cells expressing the mutant Tar (A) or
Tcp (B) protein as a sole chemoreceptor. Two microliters of fresh overnight
culture was spotted onto tryptone semisolid agar supplemented with 50 mg of
ampicillin per ml (A) or 25 mg of chloramphenicol per ml (B), and the plates
were then incubated at 30°C for 12 h. (A) Tar[NWETF] (wild type), cells carrying
pAK101; Tar[N], cells carrying pAK101-W550Op; None, cells carrying pBR322.
(B) Tcp[NWESF], (wild type), cells carrying pAS101; Tcp[NWESLA], cells carrying pAS103; Tcp[NWES], cells carrying pAS104; Tcp[NWESC], cells carrying
pAS105; Tcp[NWESA], cells carrying pOKU106; None, cells carrying pSU21.

into strain HCB399. The transformants were tested for theirability to swarm in tryptone semisolid agar (0.3%). Cells expressing the mutant Tar protein spread much slower thanthose expressing wild-type Tar (Fig. 2A). Similarly, cells expressing any mutant Tcp spread slower than cells expressing
wild-type Tcp (Fig. 2B).
We then examined chemotactic responses directly by a temporal stimulation assay. The SSF of HCB339 cells expressing
wild-type Tar was about 70% without chemotactic stimuli (Fig.
3A and B). When the repellent 10% glycerol was added, theSSF decreased to almost nothing, a response that persisted forat least 8 min. Immediately after 10 or 100 mM aspartate wasadded to cells tumbling in response to glycerol, the SSF
jumped to 80% but then began to decrease gradually (Fig. 3A
and B). This gradual decrease in SSF was not observed for cells
expressing mutant Tar. They gave a persistent SSF in response
to aspartate, demonstrating that the mutant Tar is defective in
adaptation (Fig. 3C and D).
The SSF of cells expressing wild-type Tcp was about 80%
without stimulation (Fig. 4). The addition of 10% glycerol
reduced the SSF to almost nothing, but the SSF increased
gradually to 40% after 7 min. Immediately after 10 mM citrate
was added, the SSF increased to almost 100% and then decreased gradually to reach about 60% after 7 min. By contrast,
cells expressing mutant Tcp proteins produced little or nochange in the SSF after the initial response to glycerol orcitrate (Fig. 4B to E). These results suggest that mutations in
the C-terminal pentapeptide sequence of Tcp resulted in impaired adaptation after both attractant and repellent responses.
Attractant-sensing abilities and expression levels of mutant
Tar and Tcp proteins. Impaired adaptation in the mutants
might be an indirect result of an increased affinity to a ligand
or increased signaling strength, either of which could mask
adaptation. We therefore examined the dose-response characteristics of the mutant Tar (Fig. 5) and Tcp (data not shown)proteins. The swimming behavior of cells expressing each mutant receptor was observed just after the addition of various
concentrations of an attractant. When 10% glycerol was added,
the SSF of cells expressing either wild-type or mutant receptor

FIG. 3. Adaptation of HCB339 cells expressing the mutant Tar protein. At
the time indicated by the first downward arrow, glycerol was added to 10% ({and h), and at the time indicated by the second downward arrow, aspartate wasadded at 10 (A and C) or 100 (B and D) mM (h). (A and B) Tar[NWETF] (wildtype), cells carrying pAK101; (C and D) Tar[N], cells carrying pAK101-W550Op.

was insignificant. Cells expressing wild-type Tar began to respond to 1027 M aspartate and gave a maximum response at1025 M or higher concentrations of aspartate (Fig. 5A). The
mutant Tar had a slightly weaker apparent affinity for aspartate
than wild-type Tar (Fig. 5B). Cells expressing wild-type Tcp
began to respond at 1025 M citrate and showed a maximum
response at 1023 M or higher concentrations of citrate (datanot shown). The concentrations of citrate required for attractant responses mediated by mutant Tcp proteins were similar toor slightly higher than those for wild-type Tcp (data notshown). These results indicate that the mutations in the Cterminal sequences did not increase ligand-binding affinity or
signaling.
We also examined the expression levels of the mutant receptors by immunoblotting with anti-Tsr serum (17), whichcross-reacts with both Tar and Tcp. Strain CP553, which lacks
the methyltransferase CheR, the methylesterase CheB, and thefour chemoreceptors, was used as the plasmid host. Receptor
proteins in this strain exist as single species because of theabsence of posttranslational methylation or deamidation, acircumstance that makes comparison of their expression levels
easier. Tar[N] was expressed at almost the same level as wildtype Tar (Fig. 6A). Tcp[NWESLA] and Tcp[NWESC] appeared to form slightly more intense and slightly less intense
bands, respectively, than wild-type Tcp (Fig. 6B). The bands
formed by all of the other mutant Tcp proteins were of almost
the same intensity as that of wild-type Tcp (Fig. 6B; data for
Tcp[NWESA] not shown). These results suggest that the mutations in the C-terminal sequences did not dramatically affectsynthesis or degradation of the receptor proteins.
Methylation patterns of mutant Tar and Tcp proteins.
Methylation levels in the CheB1 CheR1 background weremonitored by immunoblotting. Methylation of a receptor increases its mobility in SDS-polyacrylamide gel electrophoresis

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FIG. 5. Dose-response relationship for the wild-type and mutant Tar proteins. HCB339 cells expressing the wild-type or mutant receptor were pretreatedwith 10% glycerol to reduce the SSF and were then exposed to various concentrations of aspartate. The SSF was measured within 1 min. (A) Tar[NWETF](wild type), cells carrying pAK101; (B) Tar[N], cells carrying pAK101-W550Op.

FIG. 4. Adaptation of HCB339 cells expressing each mutant Tcp protein.
Assays were carried out as described in the legend to Fig. 3, using 1 mM citrateinstead of aspartate. (A) Tcp[NWESF] (wild type), cells carrying pAS101; (B)Tcp[NWESLA], cells carrying pAS103; (C) Tcp[NWES], cells carrying pAS104;(D) Tcp[NWESC], cells carrying pAS105; (E) Tcp[NWESA], cells carrying
pOKU106.

(5, 7, 8, 10). In the absence of aspartate, the methylation levelof the mutant Tar protein was comparable to that of wild-type
Tar (Fig. 6C). However, methylation of the mutant Tar was
stimulated only slightly by aspartate. In the case of Tcp (Fig.
6D), all of the mutant proteins were significantly less methylated than wild-type Tcp in the absence of any chemoeffector(i.e., upper bands appeared). This seemed specific to Tcp,
because the unstimulated methylation level of Tar[NWETA]
was similar to that of Tar[N] (unpublished results). Their
methylation levels increased little or only slightly when cells
were incubated with citrate (NWESLA . NWES .
NWESC ' NWESA). These results suggest that the mutationsin the C-terminal sequence severely affect the attractant-stimulated methylation.
Suppression of the defect in adaptation and methylation ofmutant Tar by overproduction of CheR. While this work was inprogress, in vitro results demonstrating that CheR binds to the
NWETF sequence of Tsr were published (53). If the C-terminally-mutated receptors have weaker affinities to CheR, theirlowered methylation levels might be increased by overproduction of CheR. Alternatively, the mutant receptors could bepoor substrates for CheR, in which case overproduction of
CheR would not compensate for loss of the pentapeptide sequence. We examined these possibilities with the mutant Tar
protein.
We examined whether CheR overproduction suppresses thedefect in adaptation of the mutant Tar protein. Cells expressing mutant Tar were transformed with a CheR-expressing(pRAR1) or a CheR-overproducing (pKB23) plasmid. In both
tryptone and aspartate-minimal semisolid agar, the swarming
ability of HCB339 cells expressing the mutant Tar protein wasenhanced by overproduction of CheR from the induced tac
promoter (Fig. 7A and B; compare results for Tar[N] 1
CheR1 and Tar[N] 1 CheR111). The resulting chemotactic
rings were of comparable size to those of cells expressing
wild-type Tar and containing the wild-type level of CheR (Fig.
7A and B; compare results for Tar[N] 1 CheR111 and Tar[NWETF] 1 CheR1).
Adaptation times also were determined for cells overproducing CheR (Fig. 7C and D). Without any chemoeffector, the
SSF of strain HCB339 expressing mutant Tar and overproducing CheR was only about 20%, although the resting methylation level was not much changed (see below). The reason forthis reduced SSF is not clear, but one possibility may be thatsome stress results from carrying two different multicopy plasmids. The SSF reached 60 and 80% immediately after the
addition of 10 and 100 mM aspartate, respectively, and thendecreased gradually. These results suggest that the defect inadaptation in the mutant was suppressed by CheR overproduction.
Receptor methylation was then monitored by immunoblotting (Fig. 6C). With increasing levels of expression of CheR,
the receptor became more methylated in the presence of aspartate, whereas the “resting” or unstimulated methylationlevel did not change very much. In cells carrying pKB23, in
which CheR expression was induced by 1 mM IPTG, the aspartate-stimulated methylation level of the mutant Tar wascomparable to that of wild-type Tar. These results suggest that
impaired methylation was due to decreased affinity of the mutant Tar for CheR. Therefore, the pentapeptide sequence islikely to function as a CheR-binding site in vivo, and binding of
CheR to this site is not a prerequisite for adaptive methylation.
Effects of CheR overproduction on the functions of wildtype Trg. We also examined effects of CheR overproduction
on methylation and adaptation of wild-type Trg, which naturally lacks the NWETF sequence. Both in tryptone and in

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FIG. 6. Expression levels (A and B) and methylation patterns (C and D) of
the mutant Tar (A and C) and Tcp (B and D) proteins. (A and B) The wild-type
or mutant receptors were expressed in the CheB2 CheR2 strain CP553 anddetected by immunoblotting with anti-Tsr serum. Tar[NWETF] (wild type), cells
carrying pAK101; Tar[N], cells carrying pAK101-W550Op; Tcp[NWESF] (wildtype), cells carrying pAS101; Tcp[NWESLA], cells carrying pAS103; Tcp[NWES], cells carrying pAS104; Tcp[NWESC], cells carrying pAS105; None,
cells carrying pSU21. (C and D) Cells expressing each receptor were preincubated with distilled deionized water (lanes D), 10 mM aspartate (lanes A), 10mM citrate (lanes C), or 10% glycerol (lanes G). Chemoreceptors were detected
as described for panels A and B. Tar[NWETF] (EEEE) CheB2 CheR2, CP553
cells carrying pNI130; Tar[NWETF], HCB339 cells carrying pAK101; Tar[N],HCB339 cells carrying pAK101-W550Op; Tar[N] 1 CheR11, HCB339 cells
carrying pAK101-W550Op and pRAR1 (a multicopy plasmid bearing cheR);
Tar[N] 1 CheR111, HCB339 cells carrying pAK101-W550Op and pKB23 (aCheR2 overproducer plasmid), induced for CheR expression with 1 mM IPTG;
Tcp[NWESF] CheB2 CheR2, CP553 cells carrying pAS101; Tcp[NWESF],
HCB339 cells carrying pAS101; Tcp[NWESLA], HCB339 cells carrying pAS103;
Tcp[NWES], HCB339 cells carrying pAS104; Tcp[NWESC], HCB339 cells carrying pAS105; Tcp[NWESA], HCB339 cells carrying pOKU106.

ribose-minimal semisolid agar, cells expressing Trg as solechemoreceptors swarmed better when CheR was overproduced from the tac promoter than when CheR was not
overproduced (Fig. 8A and B). Overproduction of CheR
enhanced stimulation of methylation by the Trg-specific attractant ribose and slightly increased methylation of unstimulated Trg (Fig. 8C). These results suggest that the
weak adaptation and methylation phenotypes of wild-type
Trg are mainly due to the lack of the primary CheR-binding
site, a deficit which can be overcome by CheR overproduction.

J. BACTERIOL.

FIG. 7. Effects of CheR overproduction on swarming (A and B) and adaptation (C and D) supported by mutant Tar. (A and B) Two microliters of fresh
overnight culture of HCB339 cells carrying Tar-expressing and CheR-expressing
plasmids was spotted onto tryptone semisolid agar or minimal semisolid agar
containing 100 mM aspartate supplemented with ampicillin, chloramphenicol,
and IPTG, and the plates were incubated at 30°C for 10 (A) or 18 (B) h.
Receptor proteins and the plasmids bearing the genes encoding them: Tar[NWETF] (wild type), pAK101; Tar[N], pAK101-W550Op; CheR expression
levels and the plasmid introduced: CheR1 (wild-type level), pACYC184 (vector); CheR11 (overproduced), pRAR1 (a multicopy plasmid bearing cheR);
CheR111 (highly overproduced), pKB23 (a CheR-overproducing plasmid). (Cand D) Chemotactic responses to 10 (C) or 100 (D) mM aspartate were measured as described in the legend to Fig. 3 but without pretreatment with glycerol.

DISCUSSION
We here investigated the effects of mutations altering the
C-terminal NWET/SF sequence which is conserved among the
major chemoreceptors of E. coli and S. typhimurium (Tsr, Tar,and Tcp) but not among the minor chemoreceptors (Trg andTap). The recent in vitro study showed that the NWETF sequence of Tsr is a primary site for CheR binding (53). Consistent with this, mutations in Tar and Tcp impaired their adaptation and methylation abilities without much affecting theircellular levels and apparent ligand-binding affinities. Overproduction of CheR suppressed the adaptation- and methylationdefective phenotypes of the mutant Tar protein as well as thoseof wild-type Trg. These in vivo results demonstrate that the
NWET/SF sequence is not a prerequisite for receptor methylation itself but suggest that the sequence facilitates effective
adaptive methylation by recruiting CheR.
Although aspartate-stimulated methylation of Tar[N] wasrestored by overproduction of CheR, its unstimulated methylation level was almost the same as that of cells expressing thenormal level of chromosomally encoded CheR (Fig. 6C). Thus,

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other receptor dimer as discussed above). Alternatively, the
NWETF sequence might exist only to provide a docking site
for CheR. Recruitment of CheR to the receptor will increase
the local concentration of the enzyme and should thereby
increase the probability of its collision with the methylationsites.
In any case, a multiprotein complex containing the receptor,
CheA, CheW, and CheR clearly plays a central role in chemotactic signal transduction. Schuster et al. (40) showed that
CheY binds stably to the receptor-CheW-CheA ternary complex and that CheY is released from the complex when phosphorylated. It is reasonable to speculate that CheB behaves ina similar fashion. Thus, the whole chemotactic signal transduction system is apt to be spatially well organized rather than toconsist of freely diffusible components encountering eachother stochastically.
Such higher-order structures have been implicated in eukaryotic signal transduction. For example, the yeast STE5 protein serves as a scaffold for members of the mitogen-activatedprotein kinase cascade, and the mammalian A-kinase anchoring protein AKAP79 tethers the type II cyclic AMP-dependent
protein kinase, protein kinase C, and calcineurin to thepostsynaptic densities (11). These complexes may guarantee
rapid and discrete flows of information by placing their components in the correct sequence to promote signal transduction
and to prevent cross talk between homologous but distinct
systems.
FIG. 8. Effects of CheR overproduction on swarming (A and B) and methylation (C) supported by wild-type Trg. HCB339 cells carrying pCP31 (trg1) weretransformed with plasmid pACYC184 (vector; CheR1), pRAR1 (a multicopy
plasmid bearing cheR; CheR11), or pKB23 (a CheR-overproducing plasmid;
CheR111). (A and B) Swarming was assayed as described in the legend to Fig.
7, using 10 mM ribose instead of aspartate. (C) Receptor methylation was
assayed as described in the legend to Fig. 6, using 10 mM ribose (R) as an
attractant. Lanes labeled D and G are defined in the legend to Fig. 6.

the NWETF sequence might be responsible for allowing thehigh initial rate of methylation upon stimulation with an attractant but dispensable for the resting steady-state level ofmethylation. The situation is similar for wild-type Trg, but its
unstimulated methylation levels increased slightly with increasing levels of CheR (Fig. 8A). This difference might be due toeither the absence of more of the carboxy-terminal sequence ofthe major receptors from wild-type Trg (Fig. 1) than fromTar[N] or to other intrinsic differences between Trg and Tar.
Wu et al. (53) proposed that CheR bound to a receptor
dimer may catalyze methylation of a neighboring receptor
dimer. This interdimer methylation model can explain why Trg
is more efficiently methylated when Tsr and/or Tar is present
than when Trg is expressed alone (20, 55). The possibility ofinterdimer interaction in signal production has also been argued, since Tar dimers with only one intact cytoplasmic domain have attractant signaling abilities (13, 49). Although these
possibilities remain to be tested experimentally, it is quitepossible that receptor dimers encounter each other in thecytoplasmic membrane, because the receptors have beenshown to cluster at the poles of E. coli cells (27).
Unlike the situation with CheB, there is no evidence that theactivity of CheR is regulated. Identification of a CheR-bindingsite on the major receptors distinct from their methylation sites
suggests the following possible mechanisms for regulation of
CheR activity, however: (i) CheR might associate with thereceptor under some conditions and dissociate from it underothers; (ii) ligand binding might change the configuration ofbound CheR relative to the methylation sites (possibly in an-

ACKNOWLEDGMENTS
We thank G. L. Hazelbauer, K. Oosawa, and M. I. Simon for providing us with plasmids. We especially thank M. D. Manson of Texas
A & M University for critically reading the manuscript.
This work was supported in part by a grant-in-aid for scientificresearch to I.K. from the Ministry of Education, Science, and Cultureof Japan.

ADDENDUM IN PROOF
Recently, Li et al. (J. Li, G. Li, and R. M. Weis, Biochemistry 36:11851–11857, 1997) and Le Moual and Koshland (H.
Le Moual and D. E. Koshland, Jr., Biochemistry 36:13441–
13448, 1997) demonstrated interdimer methylation by in vitro
assays.
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