JOURNAL OF BACTERIOLOGY, Nov. 2005, p. 7647–7654
0021-9193/05/$08.00⫹0 doi:10.1128/JB.187.22.7647–7654.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 187, No. 22

Stabilization of Polar Localization of a Chemoreceptor via Its Covalent
Modifications and Its Communication with a Different Chemoreceptor
Daisuke Shiomi,1† Satomi Banno,1 Michio Homma,1 and Ikuro Kawagishi1,2*
Division of Biological Science, Graduate School of Science,1 and Institute for Advanced Research,2 Nagoya University,
Chikusa-ku, Nagoya 464-8602, Japan
Received 12 January 2005/Accepted 26 July 2005

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, andthe adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, themechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we
examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor
Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced
its own polar localization, although the effect was small. The slight but significant effect of amidation on
receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR
that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation.
Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine
chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small toaccount for chemotactic adaptation, but receptor modification is suggested to contribute to the molecularassembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor
dimer-to-dimer interaction.
facilitated by receptor amidation, which is equivalent to methylation (18, 21). Second, unlike the high-abundance receptors(Tsr and Tar), the low-abundance receptors (Tap and Trg) are
not methylated effectively when expressed as the sole chemoreceptors (41) and localize to a cell pole but do not cluster with
CheA and CheW (23). Third, recent fluorescence resonanceenergy transfer (FRET) analyses have demonstrated that receptor methylation decreases sensitivity to an attractant, presumably by cooperative interactions of receptor dimers (35,36).
Several independent lines of evidence suggest that chemoreceptor dimers interact with each other in vivo. A cytoplasmic
fragment of Tsr crystallizes with a unit of a “trimer of dimers”
through the interaction at its cytoplasmic highly conserved
domain (HCD) (13, 14), which is conserved among all of thechemoreceptor proteins (17, 42). Based on crystallography, it is
proposed that chemoreceptors form a hexagonal network (14,31). Studies with mutagenesis, cross-linking, and immunoprecipitation showed that Tar and Tsr interact with each otherthrough the HCD in vivo (1). More recently, in vivo experiments with trifunctional cross-linking reagents have strongly
suggested that mixed trimers of Tar and Tsr dimers exist in thecell under normal conditions (37). Studies with chemically synthesized multivalent ligands suggested that an attractant response is amplified by the interaction between dimers of thesame and different chemoreceptor species (8, 16). It was recently demonstrated by FRET analyses that receptor cooperativity is affected by the expression level of a chemoreceptor
and the existence of other chemoreceptors (36).
In this study, we found that the polar localization of Tar
fused to green fluorescent protein (GFP) is influenced by its
own amidation (methylation) state and the expression of another chemoreceptor (Tsr). Covalent modification is suggested
to stabilize polar localization of the chemoreceptors, presum-

Spatial regulation of the subcellular localization of proteinsis important for various cellular events in both prokaryotic andeukaryotic cells. In prokaryotes, for example, polar protein
localization has been implicated in cell division, virulence, andchemotaxis (24, 31). In the chemotaxis of Escherichia coli, a setof transmembrane receptors named chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), together with thehistidine kinase CheA and the adaptor CheW, cluster at a cellpole (26, 34). This polar clustering of the chemotactic machinery is thought to be required for normal signal amplification
and adaptation (1, 4, 8, 32, 36).
E. coli has four chemoreceptors (Tsr for serine, Tar for
aspartate and maltose, Tap for dipeptides, and Trg for ribose
and galactose) and one MCP-related protein involved in redox
taxis (Aer). A chemoreceptor forms a homodimer regardlessof its ligand occupancy state (27). Other chemotaxis signaling
proteins (i.e., CheY, which controls the rotational sense of theflagellar motor, and CheZ, which facilitates dephosphorylationof CheY, the methyltransferase CheR, and the methylesterase
CheB) also target to the receptor-kinase cluster (2, 5, 33, 34).
However, despite growing knowledge of the three-dimensional
structures of and interactions between the signaling components, the mechanism that leads to the polar localization of thereceptor is still largely unknown.
Receptor methylation, a key process of adaptation, might bea good candidate for a factor affecting the localization of chemoreceptors. First, the formation of an in vitro complex consisting of a cytoplasmic fragment of Tar, CheA, and CheW is* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku,
Nagoya 464-8602, Japan. Phone: 81 52 789 2993. Fax: 81 52 789 3001.
E-mail: i45406a@cc.nagoya-u.ac.jp.
† Present address: Microbiology and Molecular Genetics, Universityof Texas Medical School, Houston, TX 77030.
7647

7648

SHIOMI ET AL.

J. BACTERIOL.

ably through interdimer interactions. The greater tendency ofamidated forms of the receptors to localize to the cell pole may
be critical in biogenesis of the polar clusters and may provide
an explanation for why the receptors are initially translated in
the half-amidated forms rather than the nonmodified (all-glutamate) forms.
MATERIALS AND METHODS
Bacterial strains and plasmids. All strains used in this study are derivative ofE. coli K-12. Strain RP437 is wild type for chemotaxis (29). Strains HCB339 (39),
HCB436 (40), and HCB437 (40) lack all four chemoreceptors (MCPs). In addition, strain HCB436 lacks CheB and CheR and strain HCB437 lacks all of the
Che proteins. Strain AW518 lacks Tsr (15).
The vector plasmids pBAD24 (carrying the ampicillin resistance gene [Apr])and pBAD33 (carrying the chloramphenicol resistance gene [Cmr]) (9) carry thearaBAD promoter and the araC gene, which encodes the positive and negative
regulator of the araBAD promoter. Plasmid pEGFP, which encodes enhanced
GFP, and plasmid pTrcHisB, which carries the trc promoter, the lacIq gene, andthe bla gene, were purchased from Clontech and Invitrogen, respectively.
The pBAD24-based plasmid pDS223 carries GFP-CheR-D154A (33). The
methylation sites of Tar-QEQE on pLC113 plasmids were mutagenized to yield
pDS1000 (Tar-EEEE) and pDS1014 (Tar-QQQQ), respectively.
Construction of the plasmids encoding Tar-GFP. The PstI-HindIII fragmentof pDS1020, carrying the gene for Tar-QEQE-GFP placed downstream of the trc
promoter (10), was cloned into the PstI-HindIII sites of pHS401 (H. Sakamotoand I. Kawagishi, unpublished), carrying the gene for Tar placed downstream of
the araBAD promoter, to yield the plasmid encoding Tar-QEQE-GFP under thecontrol of the araBAD promoter (named pDS1030). The NdeI-PvuII fragmentsof pDS1000 (encoding Tar-EEEE) and pDS1014 (encoding Tar-QQQQ) werecloned between the corresponding sites of pDS1030 to yield the plasmids encoding Tar-EEEE-GFP (named pDS1031) and Tar-QQQQ-GFP (named
pDS1032), respectively. The PstI-HindIII fragments of pDS1031 and pDS1032
were cloned into the corresponding region of pDS1020 to yield the plasmids
encoding Tar-EEEE-GFP (named pDS1021) and Tar-QQQQ-GFP (named
pDS1022) under the control of the trc promoter, respectively.
Immunoblotting. Receptor methylation and expression levels of proteins weremonitored by immunoblotting as described previously (28) with anti-Tsr serum(11), which cross-reacts with Tar, or anti-GFP antibody (Molecular Probes). The
first antibodies were detected with alkaline phosphatase-conjugated anti-rabbit
immunoglobulin G (Vector Laboratories) or horseradish peroxidase-conjugatedanti-rabbit immunoglobulin G (New England BioLabs).
Fluorescence microscopy. Observations of fluorescence of Tar-GFP or GFPCheR were carried out essentially as described previously (2, 33). The culture
conditions were similar to those used for the immunoblotting analyses. Cells
were harvested, washed twice with MLM (10 mM potassium phosphate buffer[pH 7.0], 0.1 mM EDTA, 10 mM DL-lactate, 0.1 mM methionine), and resuspended in MLM medium. In order to examine the effects of Tsr on the localization of Tar-GFP, HCB436 (CheA⫹ CheW⫹) cells were transformed withplasmid pDS1021 (encoding Tar-EEEE-GFP) and then further transformed withplasmid pBAD33 or pOH351 (tsr⫹; Motohiro Homma and Ikuro Kawagishi,
unpublished results). The transformants were grown in TG medium (1% tryptone, 0.5% NaCl, 0.5% [wt/vol] glycerol) supplemented with 50 ␮g/ml ampicillinand 25 ␮g/ml chloramphenicol with vigorous shaking at 30°C. After 3 h, various
concentrations of arabinose (for expression of Tsr) were added to the cultures.
Cells were grown further for 0.5 h, harvested, washed twice with MLM, andresuspended in MLM medium. Small aliquots of the cell suspensions werespotted onto glass slides coated with 0.5% agarose and were observed under aninverted fluorescence microscope IX70 (Olympus). The images were recorded
and processed by using cooled charge-coupled-device camera CoolNAP-FX/OL(Roper) and the software Meta Morph version 5.0r4 (Roper).

RESULTS
Construction and characterization of Tar-GFP. To investigate factors which affect the polar localization of the chemoreceptor (MCP), we first tested the effect of methylation statesof the aspartate chemoreceptor (Tar) on its own polar localization. Nascent Tar has four potential methylation sites:
Q295, E302, Q309, and E491, collectively referred to as QEQE

FIG. 1. Construction and characterization of Tar-GFP. (A) Schematic illustration of Tar-GFP. Open circles indicate four methylationsites (Q295, E302, Q309, and E491). Filled and shaded boxes represent
two transmembrane domains (TM1 and TM2) of Tar and GFP, respectively. (B) Swarming abilities of cells expressing Tar-QEQE-GFP.
AW518 (tsr) was used as a control. Shown is the swarming of HCB339(⌬MCP) cells carrying control vector or the plasmid encoding TarQEQE-GFP. Cells were inoculated onto a semisolid agar plate (0.3%)
and incubated at 30°C for 12 h. (C) Expression levels of Tar-GFPs in
strains HCB436 (⌬MCP CheA⫹ CheW⫹) and HCB437 (⌬MCPCheA⫺ CheW⫺). E, Tar-EEEE-GFP; QE, Tar-QEQE-GFP; Q, TarQQQQ-GFP.

(Fig. 1A). The two glutamine residues (Q) are deamidated by
CheB to become methylatable glutamate residues (E). A glutamine residue mimics methylated glutamate residue (Em) (3,6), and therefore Tar-QQQQ represents the fully methylated
form of Tar. To examine the effect of receptor methylation, wetherefore introduced the EEEE and QQQQ configurations
into Tar-GFP. In the swarm assay using tryptone semisolid
agar (0.3%), HCB339 (⌬MCP) cells expressing the TarQEQE-GFP protein (10) formed a swarm ring (Fig. 1B). Although the level of functionality cannot be quantified fromthese plates, HCB339 cells expressing the GFP fusion protein
swarmed only slightly slower than those expressing the intact
Tar protein (data not shown), indicating that the GFP construct retains essential receptor function. All of the Tar-GFP
proteins were expressed in strains HCB436 (⌬MCP ⌬CheRB;
hereafter referred to as CheA⫹ CheW⫹) and HCB437 (⌬MCP⌬CheAWRBZY; hereafter referred to as CheA⫺ CheW⫺).
Immunoblotting analyses revealed that their expression levels
were comparable (Fig. 1C) and did not exceed the total
amount of chromosome-encoded Tar and Tsr (data notshown).
Effect of covalent modification on polar localization of TarGFP. Tar-QEQE-GFP localized to cell poles in the presence of

VOL. 187, 2005

STABILIZATION OF CHEMORECEPTOR LOCALIZATION

7649

FIG. 2. Effect of covalent modification of localization of Tar-GFP. Shown are the subcellular localizations of the variously amidated versionsof Tar-GFP in strains HCB436 (⌬MCP CheA⫹ CheW⫹; upper) and HCB437 (⌬MCP CheA⫺ CheW⫺; lower). EEEE, Tar-EEEE-GFP (left);
QEQE, Tar-QEQE-GFP (middle); QQQQ, Tar-QQQQ-GFP (right).

CheA and CheW, but the extent of its polar localization was
reduced substantially in the absence of CheA and CheW (10)(Fig. 2). Microscopic observation detected no obvious difference in the localization among the variously amidated versionsof Tar-GFP in the CheA⫹ CheW⫹ background (strain
HCB436) (Fig. 2). By contrast, in the CheA⫺ CheW⫺ background (strain HCB437), Tar-QQQQ-GFP localized much
more effectively to cell poles than Tar-EEEE-GFP (Fig. 2).
To quantify the effect of covalent modification on localization, images of cells were recorded, and the fluorescence intensity was quantitated by scanning the image in the directionof the major (length) and minor (width) axes for each cell (Fig.
3A). The maximum intensity of all scans was subsaturating andtherefore quantifiable. The difference between the peak andthe basal lines was defined as “intensity.” Forty HCB437(CheA⫺ CheW⫺) or HCB436 (CheA⫹ CheW⫹) cells expressing either the EEEE or QQQQ version of Tar-GFP were
examined, and the results are presented in histograms (Fig.
3B). In the CheA⫺ CheW⫺ strain, the QQQQ version more
effectively localized in the major axis than did the EEEE version. These quantitative results reinforce the qualitative observations made by eye as described above. Even in the CheA⫹
CheW⫹ strain, the distribution of major-axis fluorescence intensity of the amidated version was slightly but significantly
shifted to the higher values (i.e., to the right in the histogram)compared with that of the demethylated/deamidated one (major axis), suggesting that the methylation (amidation) level of
Tar influences its own subcellular localization even in the wildtype background.
Effect of receptor covalent modification on polar localization of GFP-CheR. Because the effect of covalent modification
described above was subtle, especially in the presence of CheA
and CheW, and localization of Tar-GFP does not exactly reflect that of Tar itself (2), we wanted to examine whether
localization of the nonfusion chemoreceptor is also influenced

by its own methylation. Since CheR targets to the NWETF
sequence (33) and there is no known regulation of this interaction, its localization should reflect that of the chemoreceptor. We therefore examined the effect of receptor methylation
on localization of GFP-CheR. In this assay, we used a mutant
version (D154A) of GFP-CheR that lacks enzymatic activity toalter the methylation state of the receptor (33); otherwise, theEEEE receptor would have been methylated. We previously
showed that the mutation D154A does not impair a polartargeting of GFP-CheR (33). HCB436 cells (CheA⫹ CheW⫹)
that lack CheR and CheB as well as all four chemoreceptors
were transformed first with a plasmid encoding Tar-EEEE(pDS1000) or Tar-QQQQ (pDS1014) and then with a plasmid
encoding GFP-CheR-D154A. For each amidation state, morethan 500 cells were observed, and the fraction of cells withpolar fluorescent spots was scored. The observations were triplicated, and the average values of the polar fraction are shownwith standard deviations in Fig. 4. GFP-CheR showed slightly
but significantly more polar bias when coexpressed with the
QQQQ receptor than with the EEEE receptor (Fig. 4), supporting the notion that receptor methylation affects polar localization of the receptor itself.
Communication between two different chemoreceptors. It
has been reported that dimers of the same or different chemoreceptor species interact with each other (1, 8, 16). This
prompted us to examine whether the interaction between different chemoreceptor dimers in vivo is also involved in theirpolar localization. HCB436 (CheA⫹ CheW⫹) cells expressing
Tar-EEEE-GFP were transformed with plasmid pBAD33(vector) or pOH351 (pBAD33-tsr). Immunoblotting analyses
showed that increasing concentrations of arabinose resulted inincreasing levels of Tsr but not of Tar-EEEE-GFP (Fig. 5). We
observed subcellular localization of Tar-EEEE-GFP in thepresence of various concentrations of arabinose (Fig. 6A and
B). As the expression level of Tsr increased, the extent of polar

7650

SHIOMI ET AL.

J. BACTERIOL.

FIG. 3. Quantitative analysis of the effect of covalent modification on receptor localization. (A) Schematic illustration of the quantification
procedure. Each cell was scanned for fluorescence intensity in its major and minor axes with the line scan mode of the image-analyzing software(MetaMorph version 5.0r4). The “fluorescence intensity” of a cell in one axis was defined as the difference between the highest and the lowest
values (in arbitrary units) within the cell, as indicated in the diagram. (B) Histograms of fluorescence intensities of 40 HCB437 (⌬MCP CheA⫺
CheW⫺; upper) or HCB436 (⌬MCP CheA⫹ CheW⫹; lower) cells expressing the EEEE (open bars) or QQQQ (filled bars) version of Tar-GFP
in major (left) and minor (right) axes. Three independent experiments were carried out, and representative data are shown.

localization of Tar-EEEE-GFP appeared to increase. We carried out semiquantitative analysis of this effect (Fig. 6C). Observed cells were divided into three classes in terms of thelocalization of fluorescence: completely polar, polar and lateral, and nonpolar. With increasing levels of Tsr, the numbersof nonpolar and completely polar cells decreased and increased, respectively. In the presence of 200 ␮M arabinose, theproportion of completely polar cells reached a maximum,
whereas a higher concentration (2 mM) of arabinose decreased the number of completely polar cells and increased thenumber of polar and lateral cells (see the Discussion). Even in
HCB437 (CheA⫺ CheW⫺) cells, increasing levels of Tsr promoted polar localization of Tar-EEEE-GFP (Fig. 6B). These
results suggest that a chemoreceptor can recruit a different
chemoreceptor species to a pole, presumably by coclustering.

DISCUSSION
In this study, we found factors stabilizing the polar localization of the aspartate chemoreceptor (MCP) Tar using its GFP
fusion derivative. It was previously shown that the polar localization of chemoreceptors does not require either CheR or
CheB (22). However, this does not necessarily mean that covalent modification has no effect on receptor localization. Evenin ⌬cheR and ⌬cheB strains, chemoreceptors were not fully
deamidated and methylated, respectively (data not shown),
and therefore these strains might contain chemoreceptors withvarious methylation/amidation states. Consequently, the difference in methylation levels between the two strains might not bevery large. Moreover, when another amidated receptor (Tsr) is
coexpressed, Tar-EEEE-GFP could be localized effectively toVOL. 187, 2005

STABILIZATION OF CHEMORECEPTOR LOCALIZATION

7651

FIG. 5. Coexpression of Tsr with Tar-EEEE-GFP. Expression levels of Tar-EEEE-GFP and Tsr in HCB436 (⌬MCP CheA⫹ CheW⫹)
cells were detected with anti-Tsr, which cross-reacts with Tar. To
induce the expression of Tsr, arabinose was added to cell culture at theindicated concentrations. The same cultures were used in microscopic
observation (Fig. 6).

FIG. 4. Effect of receptor methylation on subcellular localization of
GFP-CheR that lacks catalytic activity. The GFP-CheR fusion proteinwith a substitution (D154A) at the catalytic center of CheR was coexpressed with the EEEE and QQQQ forms of Tar in HCB436 (⌬MCPCheA⫹ CheW⫹) cells in the presence of 0.5 mM arabinose (for GFPCheR) and 0.1 ␮M sodium salicylate (for Tar). (A) Fluorescence
images. (B) Fraction of cells with polar fluorescent spots. More than
500 cells were scored for each condition. The observations were triplicated, and the average values of the polar fraction are shown withstandard deviations. Note that no polar spot was observed for HCB436
cells expressing GFP-CheR without any receptor (not shown, but see
reference 33).

cell poles, as shown in Fig. 6. A more recent immunoelectron
microscopic study by Lybarger et al. (25) showed that localization of low-abundance chemoreceptors, but not high-abundance ones, is facilitated by amidation. Their results are partially inconsistent with ours. We found that amidation slightly
but significantly enhances localization of the GFP fusion to thehigh-abundance chemoreceptor (i.e., Tar). This does not seemto be an artifact, because the localization of GFP-CheR, whichtargets to the high-abundance chemoreceptors, was also enhanced by amidation of nonfusion Tar, presumably reflecting
the change in the localization of Tar. In fact, Liberman et al.
(20) also detected a slight decrease in localization of yellow
fluorescent protein fusions to CheA and CheZ coexpressedwith the EEEE version of Tar compared to those coexpressedwith the QEQE version. We suspect that fluorescence microscopy could pick up more subtle changes in localization thanimmunoelectron microscopy because the effect of covalentmodification on polar localization was subtle, especially in the
CheA⫹ CheW⫹ strain. In the CheA⫺ CheW⫺ strain, the difference was greater, suggesting that CheA and CheW stabilize
the polar cluster containing less-methylated forms of chemoreceptors. It should be noted that HCB437, which was used asa CheA⫺ CheW⫺ strain, is missing CheZ and CheY as well as
the MCPs CheA, CheW, CheR, and CheB. We cannot rule out
the possibility that CheZ and CheY affect the receptor localization in strain HCB436. However, it is unlikely, since theexpression of CheA and CheW can readily reverse a decreasein the interdimer cross-linking of Tar in a stain lacking all of

the Che proteins and MCPs, which reflects decreased polar
localization of Tar (10). It should also be noted that fluorescence microscopy cannot unambiguously distinguish between
polar localization and polar clustering.
An amidated cytoplasmic fragment of Tar effectively forms acomplex with CheA and CheW in vitro, whereas the deamidation of the fragment results in dissociation of the complex (18,19), suggesting that electrostatic repulsion between deamidated/demethylated methylation helices might cause the polar
cluster to be unstable. However, receptor modification states
do not affect in vivo cross-linking efficiencies at the trimer
contact interface within the cytoplasmic domain (37). In contrast, our preliminary results suggest that cross-linking of Tar
in the periplasmic domain is sensitive to its modification state(Hiroki Irieda, Motohiro Homma, and Ikuro Kawagishi, unpublished results). It is therefore reasonable to assume thatcovalent modification of a chemoreceptor may stabilize theinteraction between trimers of its dimers and/or may alter
dynamics of dimers within a trimer unit that has not been
observed in cytoplasmic cross-linking assays.
It is still unclear how chemoreceptors localize to a cell pole.
Tar-GFP, after induced, seems to appear first at lateral regionsof the membrane and later localized at a pole (D. Shiomi, M.
Yoshimoto, M. Homma, and I. Kawagishi, submitted for publication). Whatever the mechanism of migration, once Tararrives at a cell pole, it has to be captured there, as proposed
for SpoIVFB of Bacillus subtilis (30). This capture mechanism
might involve, if not strictly require, receptor clustering, sincea deletion of the cytoplasmic domain named HCD, which is
required for the interactions with neighboring dimers (to form
the “trimer of dimers” structure) and with CheA and CheW (toform the ternary complex), abolishes polar localization of TarGFP (D. Shiomi and I. Kawagishi, unpublished results). Receptor methylation and interdimer interactions may play a rolein this step. The two Gln residues (positions 295 and 309) of
Tar are posttranslationally deamidated by CheB to become
methylatable Glu residues (12). Our finding that receptor amidation influences polar localization of the receptor-kinase
complex might explain why some of the methylation sites are
encoded as Gln rather than Glu. The nascent translational
product of Tar (QEQE) is more apt to form a polar complex
with CheA and CheW than the fully deamidated form
(EEEE). If most of the CheB, CheA, and CheW molecules

7652

SHIOMI ET AL.

J. BACTERIOL.

FIG. 6. Effect of Tsr on subcellular localization of Tar-EEEE-GFP. (A and B) Fluorescence micrographs of HCB436 (⌬MCP CheA⫹ CheW⫹;
panel A) and HCB437 (⌬MCP CheA⫺ CheW⫺; panel B) cells expressing Tar-EEEE-GFP with various levels of Tsr. Arabinose was added at theindicated concentrations to induce the expression of Tsr. The same cultures were used in immunoblotting (Fig. 5). (C) Semiquantitative
characterization of the effect of Tsr on subcellular localization of Tar-EEEE-GFP in strain HCB436 (⌬MCP CheA⫹ CheW⫹). Fluorescent cells
were divided into three categories in terms of polar localization (completely polar, polar and lateral, and nonpolar). The bar graph shows thepercentages of cells belonging to these categories (filled, shaded, and open bars, respectively), with the representative micrographs above it. Three
independent experiments were carried out, and representative data are shown.

localize to the polar clusters, then chances are that a receptor
will remain in the QEQE state, and therefore in a polarly
targeted trimer of dimers, until it reaches the pole. Once incorporated into the polar receptor array, the deamidation of
the methylation sites would not remove Tar from the cluster
consisting of variously methylated/amidated forms of Tar andother chemoreceptors as well as CheA and CheW, which stabilize the polar cluster.
The coexpression of Tsr enhanced localization of Tar-GFP
to cell poles, suggesting that interaction among chemoreceptors contributes to polar localization. Consistent with this interpretation, several lines of evidence suggest directly or indi-VOL. 187, 2005

STABILIZATION OF CHEMORECEPTOR LOCALIZATION

rectly that different species of chemoreceptors can interact
with each other and cluster together (1, 8, 13, 16, 32). A
low-abundance receptor (Trg or Tap) is poorly methylated
when expressed as a sole chemoreceptor, but its methylation isenhanced by the coexpression of a high-abundance receptor(Tsr or Tar) (7, 38, 42). It was shown that Trg or Tap, when
expressed as a sole chemoreceptor, localizes to a cell pole but
does not cluster (23). Gestwicki and Kiessling (8) and Lamanna et al. (16) showed that multivalent ligands, which bindto Trg, stabilized the receptor-kinase cluster, and treatment ofcells with the multivalent ligands caused amplification of the
response to serine. Indeed, localization of low-abundance receptors is more sensitive to the modification state than that ofhigh-abundance receptors (25). In vivo cross-linking studies (1,37; Irieda et al., unpublished) showed that different types ofchemoreceptors interact with each other through their HCD.
Such a polar cluster consisting of different chemoreceptors
might be required for signal amplification and the stable polar
localization of low-abundance and/or demethylated chemoreceptors. There is a possibility that the recruitment of Tar to cell
poles by Tsr is not a Tsr-specific event. Rather, the effect might
result from “displacement” from the lateral cytoplasmic membrane because of overexpression of any membrane protein.
This possibility can be tested by examining whether the transmembrane osmosensor histidine kinase EnvZ, a GFP fusion ofwhich is distributed throughout the cytoplasmic membrane (D.
Shiomi and I. Kawagishi, unpublished results), or other membrane proteins are able to promote polar localization of the
Tar-EEEE protein.
Although Tar-GFP localized to cell poles more effectively
with increasing expression levels of Tsr, the number of completely polar cells decreased significantly when Tsr was overexpressed in the presence of 2 mM arabinose. We also observed that Tar-GFP, when overproduced, localized not only tocell poles but also to lateral cytoplasmic membranes (data notshown). At least some of these lateral populations of chemoreceptors are thought to be associated with or localized in thevicinity of the Sec protein translocation complexes (D. Shiomi,
M. Yoshimoto, M. Homma, and I. Kawagishi, submitted for
publication). The amounts of CheA and CheW could be limiting, and/or there might be no space at cell poles for too manyor too-large clusters. The in vivo molar ratio of receptor,
CheA, and CheW in a polar cluster has not been determined
precisely. It is possible that the ratio may vary under different
physiological conditions, which might be related to a variationin cooperativity that has been demonstrated by recent FRET
analyses (36).
In brief, the results obtained in this study provide snapshotsof a dynamic process of protein-protein interactions in the
chemotactic signaling system. Understanding this system indetail will require the development of probes that can monitorchanges in localization during the time course of excitation andadaptation elicited by a chemotactic stimulus.
ACKNOWLEDGMENTS
We thank Janine R. Maddock (University of Michigan) for furnishing invaluable data prior to publication and for helpful discussion,
Tomohiko J. Ito and Toshiharu Yakushi (Nagoya University) for helpful discussion and encouragement, and Howard C. Berg (Harvard
University) for critically reading the early version of the manuscript.

7653

This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (to D.S.
and I.K.).
REFERENCES
1. Ames, P., C. A. Studdert, R. H. Reiser, and J. S. Parkinson. 2002. Collaborative signaling by mixed chemoreceptor teams in Escherichia coli. Proc.
Natl. Acad. Sci. USA 99:7060–7065.
2. Banno, S., D. Shiomi, M. Homma, and I. Kawagishi. 2004. Targeting of thechemotaxis methylesterase/deamidase CheB to the polar receptor-kinase
cluster in an Escherichia coli cell. Mol. Microbiol. 53:1051–1065.
3. Borkovich, K. A., L. A. Alex, and M. I. Simon. 1992. Attenuation of sensory
receptor signaling by covalent modification. Proc. Natl. Acad. Sci. USA
89:6756–6760.
4. Bray, D., M. D. Levin, and C. J. Morton-Firth. 1998. Receptor clustering asa cellular mechanism to control sensitivity. Nature 393:85–88.
5. Cantwell, B. J., R. R. Draheim, R. B. Weart, C. Nguyen, R. C. Stewart, andM. D. Manson. 2003. CheZ phosphatase localizes to chemoreceptor patches
via CheA-short. J. Bacteriol. 185:2354–2361.
6. Dunten, P., and D. E. Koshland, Jr. 1991. Tuning the responsiveness of asensory receptor via covalent modification. J. Biol. Chem. 266:1491–1496.
7. Feng, X., J. W. Baumgartner, and G. L. Hazelbauer. 1997. High- and lowabundance chemoreceptors in Escherichia coli: differential activities associated with closely related cytoplasmic domains. J. Bacteriol. 179:6714–6720.
8. Gestwicki, J. E., and L. L. Kiessling. 2002. Inter-receptor communication
through arrays of bacterial chemoreceptors. Nature 415:81–84.
9. Guzman, L. M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J. Bacteriol. 177:4121–4130.
10. Homma, M., D. Shiomi, M. Homma, and I. Kawagishi. 2004. Attractant
binding alters arrangement of chemoreceptor dimers within its cluster at acell pole. Proc. Natl. Acad. Sci. USA 101:3462–3467.
11. Iwama, T., M. Homma, and I. Kawagishi. 1997. Uncoupling of ligandbinding affinity of the bacterial serine chemoreceptor from methylation- andtemperature-modulated signaling states. J. Biol. Chem. 272:13810–13815.
12. Kehry, M. R., M. W. Bond, M. W. Hunkapiller, and F. W. Dahlquist. 1983.
Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product. Proc. Natl. Acad. Sci. USA
80:3599–3603.
13. Kim, K. K., H. Yokota, and S.-H. Kim. 1999. Four-helical-bundle structure of
the cytoplasmic domain of a serine chemotaxis receptor. Nature 400:787–
792.
14. Kim, S.-H., W. Wang, and K. K. Kim. 2002. Dynamic and clustering modelof bacterial chemotaxis receptors: structural basis for signaling and highsensitivity. Proc. Natl. Acad. Sci. USA 99:11611–11615.
15. Kondoh, H., C. B. Ball, and J. Adler. 1979. Identification of a methylaccepting chemotaxis protein for the ribose and galactose chemoreceptors ofEscherichia coli. Proc. Natl. Acad. Sci. USA 76:260–264.
16. Lamanna, A. C., J. E. Gestwicki, L. E. Strong, S. L. Borchardt, R. M. Owen,
and L. L. Kiessling. 2002. Conserved amplification of chemotactic responses
through chemoreceptor interactions. J. Bacteriol. 184:4981–4987.
17. Le Moual, H., and D. E. Koshland, Jr. 1996. Molecular evolution of the
C-terminal cytoplasmic domain of a superfamily of bacterial receptors involved in taxis. J. Mol. Biol. 261:568–585.
18. Levit, M. N., Y. Liu, and J. B. Stock. 1999. Mechanism of CheA protein
kinase activation in receptor signaling complexes. Biochemistry 38:6651–
6658.
19. Li, G., and R. M. Weis. 2000. Covalent modification regulates ligand binding
to receptor complexes in the chemosensory system of Escherichia coli. Cell
100:357–365.
20. Liberman, L., H. C. Berg, and V. Sourjik. 2004. Effect of chemoreceptor
modification on assembly and activity of the receptor-kinase complex in
Escherichia coli. J. Bacteriol. 186:6643–6646.
21. Liu, Y., M. Levit, R. Lurz, M. G. Surette, and J. B. Stock. 1997. Receptormediated protein kinase activation and the mechanism of transmembrane
signaling in bacterial chemotaxis. EMBO J. 16:7231–7240.
22. Lybarger, S. R., and J. R. Maddock. 1999. Clustering of the chemoreceptor
complex in Escherichia coli is independent of the methyltransferase CheR
and the methylesterase CheB. J. Bacteriol. 181:5527–5529.
23. Lybarger, S. R., and J. R. Maddock. 2000. Differences in the polar clusteringof the high- and low-abundance chemoreceptors of Escherichia coli. Proc.
Natl. Acad. Sci. USA 97:8057–8062.
24. Lybarger, S. R., and J. R. Maddock. 2001. Polarity in action: asymmetricprotein localization in bacteria. J. Bacteriol. 183:3261–3267.
25. Lybarger, S. R., U. Nair, A. A. Lilly, G. L. Hazelbauer, and J. R. Maddock.
2005. Clustering requires modified methyl-accepting sites in low-abundance
but not high-abundance chemoreceptors of Escherichia coli. Mol. Microbiol.
56:1078–1086.
26. Maddock, J. R., and L. Shapiro. 1993. Polar location of the chemoreceptor
complex in the Escherichia coli cell. Science 259:1717–1723.
27. Milligan, D. L., and D. E. Koshland, Jr. 1988. Site-directed cross-linking:

7654

SHIOMI ET AL.

establishing the dimeric structure of the aspartate receptor of bacterial
chemotaxis. J. Biol. Chem. 263:6268–6275.
28. Okumura, H., S. Nishiyama, A. Sasaki, M. Homma, and I. Kawagishi. 1998.
Chemotactic adaptation is altered by changes in the C-terminal sequenceconserved among the major methyl-accepting chemoreceptors. J. Bacteriol.
180:1862–1868.
29. Parkinson, J. S., and S. E. Houts. 1982. Isolation and behavior of Escherichia
coli deletion mutants lacking chemotaxis function. J. Bacteriol. 171:6271–
6278.
30. Runder, D. Z., Q. Pan, and R. M. Losick. 2002. Evidence that subcellular
localization of a bacterial membrane protein is achieved by diffusion andcapture. Proc. Natl. Acad. Sci. USA 99:8701–8706.
31. Shapiro, L., H. H. McAdams, and R. Losick. 2002. Generating and exploiting
polarity in bacteria. Science 298:1942–1946.
32. Shimizu, T. S., N. Le Novére, M. D. Levin, A. J. Beavil, B. J. Sutton, and D.
Bray. 2000. Molecular model of a lattice of signalling proteins involved inbacterial chemotaxis. Nat. Cell Biol. 2:792–796.
33. Shiomi, D., I. B. Zhulin, M. Homma, and I. Kawagishi. 2002. Dual recognition of the bacterial chemoreceptor by chemotaxis-specific domains of the
CheR methyltransferase. J. Biol. Chem. 277:42325–42333.
34. Sourjik, V., and H. C. Berg. 2000. Localization of components of the che-J. BACTERIOL.
motaxis machinery of Escherichia coli using fluorescent protein fusions. Mol.
Microbiol. 37:740–751.
35. Sourjik, V., and H. C. Berg. 2002. Receptor sensitivity in bacterial chemotaxis. Proc. Natl. Acad. Sci. USA 99:123–127.
36. Sourjik, V., and H. C. Berg. 2004. Functional interactions between receptors
in bacterial chemotaxis. Nature 428:437–441.
37. Studdert, C. A., and J. S. Parkinson. 2004. Crosslinking snapshots of bacterial chemoreceptor squads. Proc. Natl. Acad. Sci. USA 101:2117–2122.
38. Weerasuriya, S., B. M. Schneider, and M. D. Manson. 1998. Chimeric chemoreceptors in Escherichia coli: signaling and methylation properties of
Tar-Tap and Tap-Tar hybrids. J. Bacteriol. 180:914–920.
39. Wolfe, A. J., M. P. Conley, T. J. Kramer, and H. C. Berg. 1987. Reconstitution of signaling in bacterial chemotaxis. J. Bacteriol. 169:1878–1885.
40. Wolfe, A. J., and H. C. Berg. 1989. Migration of bacteria in semisolid agar.
Proc. Natl. Acad. Sci. USA 86:6973–6977.
41. Yamamoto, K., R. M. Macnab, and Y. Imae. 1990. Repellent response
functions of the Trg and Tap chemoreceptors of Escherichia coli. J. Bacteriol.
172:383–388.
42. Zhulin, I. B. 2001. The superfamily of chemotaxis transducers: from physiology to genomics and back. Adv. Microb. Physiol. 45:157–198.