Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 200453410511063Original ArticleLocalization of receptor-modifying enzymeS. Banno, D. Shiomi, M. Homma and I. Kawagishi

Molecular Microbiology (2004) 53(4), 1051–1063

doi:10.1111/j.1365-2958.2004.04176.x

Targeting of the chemotaxis methylesterase/deamidase
CheB to the polar receptor–kinase cluster in an
Escherichia coli cell
Satomi Banno, Daisuke Shiomi,† Michio Homma andIkuro Kawagishi*
Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya
464-8602, Japan.
Summary
Chemotactic adaptation to persisting stimulation
involves reversible methylation of the chemoreceptors that form complexes with the histidine kinase
CheA at a cell pole. The methyltransferase CheR targets to the C-terminal NWETF sequence of thechemoreceptor. In contrast, localization of the methylesterase CheB is largely unknown, although regulation of its activity via phosphorylation is central toadaptation. In this study, green fluorescent protein
was fused to full-length CheB or its various parts: theN-terminal regulatory domain (N), the C-terminal catalytic domain (C) and the linker (L). The full-length and
NL fusions and, to a lesser extent, the LC fusion localized to a pole. Deletion of the P2 domain from CheA
abolished polar localization of the full-length and NL
fusions, but did not affect that of the LC fusion. Pulldown assays demonstrated that the NL fragment, but
not the LC fragment, binds to the P2 fragment of
CheA. These results indicate that binding of the NL
domain to the P2 domain targets CheB to the polarsignalling complex. The LC fusion, like the chemoreceptor, partially localized in the absence of CheA,
suggesting that the LC domain may interact with its
substrate sites, either as part of the protein or as aproteolytic fragment.
Introduction
In many sensory systems, higher order interactions
between multiple protein components are involved in signal transduction. Such interactions might be spatially reg-

Accepted 14 April, 2004. *For correspondence. E-maili45406a@cc.nagoya-u.ac.jp; Tel. (+81) 52 789 2993; Fax (+81) 52
789 3001. †Present address: Microbiology and Molecular Genetics,
University of Texas Medical School, 6431 Fannin, Houston, TX 7730,
USA.

© 2004 Blackwell Publishing Ltd

ulated. Even in bacteria, despite their small sizes, at least
some signalling components localize to subcellular compartments, and such localization seems to play criticalroles in signal transduction (Lybarger and Maddock, 2001;
Shapiro et al., 2002). Therefore, it is of vital importance toknow subcellular localization and regulated interplays
between signalling components to understand how thewhole system works to receive signals, to process information and to produce output.
The chemotactic behaviour of Escherichia coli (Manson, 1992; Stock and Surette, 1996; Falke et al., 1997;
Armitage, 1999), movement towards a more favourable
environment, is well suited to studying these issues as all
signalling components have been characterized in detail.
Intracellular chemotactic signals, which are produced bytransmembrane chemoreceptors/transducers (also known
as methyl-accepting chemotaxis proteins, MCPs) in
response to extracellular stimuli, are processed by a
His-Asp phosphorelay system (also known as a twocomponent regulatory system) that is used in a wide variety of signal transduction systems among eubacteria as
well as archaea, yeasts, fungi and higher plants (Mizuno,
1998; West and Stock, 2001). An unliganded chemoreceptor (MCP) stimulates the histidine kinase CheA, which
autophosphorylates at His-48 and donates the phosphategroup to the response regulators, CheY (at Asp-57) and
CheB (at Asp-56). Phospho-CheY binds to the flagellar
motor to induce clockwise (CW) rotation, whereas the
motor rotates counterclockwise (CCW) when phosphoCheY is not bound. Attractant binding inhibits the CheA
activity to reduce the probability of CW motor rotation andhence tumbling of the cell. Phosphorylation of the methylesterase (MEase)/deamidase (DAase) CheB enhances
its activity: it catalyses demethylation/deamidation of
MCPs. Therefore, attractant binding decreases the CheB
activity and hence promotes methylation of an MCP, whichis catalysed by the methyltransferase (MTase) CheR.
Increased receptor methylation reactivates CheA to counteract the initial response. This negative feedback regulation (termed adaptation) is essential for chemotaxis.
The cytoplasmic domain of an MCP has four or fivepotentially methylatable glutamate residues located in
two separate helices (Le Moual and Koshland, 1996;
Danielson et al., 1997); hence, the receptors are named
MCPs. The MTase CheR transfers a methyl group from1052 S. Banno, D. Shiomi, M. Homma and I. Kawagishi

Fig. 1. The GFP–CheB fusion protein and its derivatives.
A. The three-dimensional structure of Salmonella typhimurium CheB (Djordjevic et al., 1998). The N-terminal regulatory domain (N) is shown inblue, the C-terminal catalytic domain (C) in green and the linker (L) in yellow. Residues mutated in this study are indicated: Asp-11, the active
site; Asp-56, the phosphorylation site; and His-190, the catalytic site.
B. The GFP–CheB fusion protein and its derivatives constructed in this study. The residues that are mutated (D11K, D56N and H190Y) aremarked with a cross. For CheB fragments fused to GFP, residue numbers are indicated in brackets.

S-adenosyl methionine (AdoMet) to one of the glutamate
residues, and the MEase CheB hydrolyses the methylester bond of a methylated glutamate residue. Some of
the methylation sites of a newly synthesized chemoreceptor are glutamine residues that are converted to methylatable glutamate residues by the DAase activity of CheB.
CheB consists of an N-terminal regulatory domain (N) anda C-terminal catalytic domain (C) that are connected by aflexible linker (L) (Fig. 1) (Anand et al., 1998). In theunphosphorylated state, the N domain inhibits the MEase/
DAase activity by interacting with the C domain. Whenphosphorylated, CheB undergoes a conformationalchange to derepress the MEase/DAase activity (Anandet al., 1998). Unlike CheB, the MTase CheR is not regulated by covalent modification. An increase in the concentration of phospho-CheB as a result of the activation of
CheA promotes the demethylation of MCP, resulting in
adaptation. In contrast, a decrease in the concentration ofphospho-CheB causes a net increase in receptor methylation catalysed by CheR.
MCP, CheA and the adaptor protein CheW form a stable
ternary complex (Gegner et al., 1992; Schuster et al.,1993) that localizes at a cell pole (Maddock and Shapiro,
1993; Skidmore et al., 2000; Sourjik and Berg, 2000).
Such polar localization and/or clustering of the receptor–
kinase complexes has been proposed to play a critical rolein signal amplification (Bray et al., 1998; Duke and Bray,1999). The first indication of significant co-operativity, or

gain, between MCPs was provided by in vitro studies(Bornhorst and Falke, 2000; Li and Weis, 2000). In vivo,
chemically synthesized multivalent ligands induce attractant responses with lower thresholds than corresponding
monovalent ligands (Gestwicki et al., 2000; Gestwicki andKiessling, 2002). Recent analyses using fluorescence resonance energy transfer suggested that much of the gain
occurs at the receptor end of the signalling pathway (Sourjik and Berg, 2002). The cytoplasmic fragment of the serine
chemoreceptor Tsr crystallizes with a hexamer unit of atrimer of dimers (Kim et al., 1999). Such trimers of MCP
dimers have been proposed to assemble into a lattice-like
matrix (Shimizu et al., 2000; Kim et al., 2002). Genetic
analyses support the putative contacts among three
dimers, and chemical cross-linking assays detected interaction between Tar and Tsr (Ames et al., 2002). In vivo
disulphide cross-linking assays demonstrated that MCP
dimers interact with each other and suggest that attractant
binding may alter relative positions or trajectories of MCP
dimers that might form a trimer unit (Homma et al., 2004).
The receptor-modifying enzymes, CheR and CheB,
might also be recruited to the polar receptor–kinase cluster. Indeed, the b-subdomain of the MTase CheR binds to
the C-terminal pentapeptide sequence (NWETF) of highabundance MCPs (i.e. Tsr and Tar) (Wu et al., 1996;
Djordjevic and Stock, 1998; Okumura et al., 1998; Shiomi
et al., 2000), and this binding is essential for the polar
localization of CheR (Shiomi et al., 2002), increasing the© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

Localization of receptor-modifying enzyme 1053
to MCPs and raising the possibility that the methylation
helices of MCPs might serve as second targets of CheB
localization.
Results
GFP-CheB localizes to a cell pole in the presence of thechemoreceptor

Fig. 2. Swarming ability of RP4953 (DCheB) cells carrying the vector
pBAD24 (labelled none) or one of its derivatives encoding GFP(labelled GFP) or wild-type (labelled GFP–CheB), mutant (labelled
D11K, D56N or H190Y) or truncated (labelled N, NL, L, LC or C)
versions of GFP–CheB. RP437 (Che+) cells carrying the vector
pBAD24 (labelled wt) were used as a positive control. Fresh overnight
cultures (2 ml each) were spotted on tryptone semi-solid agar supplemented with 50 mg ml-1 ampicillin and 1 mM arabinose. The resulting
plate was incubated at 30∞C for 7 h.

concentration of CheR around MCPs. Barnakov et al.
(2001; 2002) reported that CheB also binds to the same
sequence albeit with much lower affinity. However, localization of the MEase/DAase CheB is largely unknown
although it plays a central role in adaptation and is alsosuggested to be involved in signal amplification (Barkai
et al., 2001; Sourjik and Berg, 2002).
In this study, we examined subcellular localization of
CheB. Observation of green fluorescence protein (GFP)
fused to CheB demonstrated that CheB localizes to a cellpole in the presence of an MCP. The GFP fusion proteinwith the N-terminal domain and the linker (NL) and, to alesser extent, that with the linker and the C-terminal
domain (LC) localized to a pole. Neither the full-length nor
the NL fusion localized to poles of cells lacking the P2
domain of CheA, whereas Tar–GFP localized normally,suggesting that CheB targets to the P2 domain of CheA.
In contrast, the LC fusion did not bind to the P2 domain
and, even in the absence of CheA, it localized partially toa pole, suggesting that the C domain by itself can target

To observe the localization of CheB, we constructed theplasmid pDS901 encoding GFP fused to wild-type CheB
(Fig. 1). Mutations were introduced into the cheB coding
region (Fig. 1): the substitution of Lys for Asp-11 (D11K)
of the regulatory domain results in a constitutive active
phenotype (pDS902); the substitution of Asn for the phosphorylated site Asp-56 (D56N) abolishes the phosphorylation of CheB (pDS904); and the substitution of Tyr for
His-190 (H190Y) of the catalytic centre eliminates the
MEase/DAase activity (pDS906) (Djordjevic et al., 1998).
RP4953 (DcheB) cells expressing wild-type GFP–CheB
swarmed in tryptone semi-solid agar slightly more slowly
than RP437 (wild-type for chemotaxis) cells (Fig. 2), indicating that the fusion protein is functional at least to considerable extent. Introduction of the H190Y mutation
abolished chemotaxis, whereas cells expressing anyother mutant version of GFP–CheB showed chemotaxis,
although they spread much more slowly than cells
expressing wild-type GFP–CheB. Immunoblotting with
anti-GFP antibody revealed that wild-type and mutant versions of GFP–CheB were expressed at similar levels
except that the H190Y mutant was expressed at a significantly lower level (Fig. 3). All the GFP–CheB proteins
were detected by antibody raised against Salmonella
typhimurium CheB (data not shown).
We then examined subcellular localization of the wildtype and mutant GFP–CheB proteins. Whereas GFP wasdistributed throughout the cell, all the GFP–CheB derivatives localized to a cell pole (Fig. 4). To avoid a relatively
high cell-to-cell variation occasionally seen for transcrip-

Fig. 3. Expression levels of wild-type, mutant
and truncated versions of GFP–CheB in
RP4953 (DCheB). GFP (pDS900) and fulllength GFP–CheB proteins [wt (wild type,
pDS901), D11K (pDS902), D56N (pDS904)
and H190Y (pDS906)] were expressed frompBAD24 derivatives with 1 mM arabinose.
The truncated versions of GFP–CheB wereexpressed, with increasing levels (denotedby triangles), from pBAD24 derivatives (N,pSB111; NL, pSB121; L, pSB131; LC, pSB141;
C, pSB151) with 1 or 5 mM arabinose or from
pTrcHisC derivatives (N, pSB211; NL, pSB221;
L, pSB231; LC, pSB241; C, pSB251) without an
inducer. These proteins were detected byimmunoblotting with anti-GFP antibody. Bands
for GFP, various versions GFP–CheB fusions
are indicated with arrowheads.
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

1054 S. Banno, D. Shiomi, M. Homma and I. Kawagishi
Fig. 4. Subcellular localization of the wild-type
and mutant versions of GFP–CheB. EachGFP–CheB fusion protein was expressed in
RP4953 (DCheB) in the presence of 1 mM
arabinose.
A. Cells expressing GFP (left, pDS900) or
GFP–CheB (right, pDS901).
B. Cells expressing mutant versions of GFP–CheB: D11K (pDS902), D56N (pDS904) andH190Y (pDS905).

tion from the arabinose promoter, GFP–CheB and its
derivatives were expressed under the control of the trc
promoter without induction, resulting in similar localization
patterns (data not shown). These results suggest that thephosphorylation and the catalytic activity of CheB are notessential for its localization.
Both N-terminal and C-terminal domains, when fused to
GFP with the linker region, can localize to a cell pole
To examine which region of CheB is responsible for thepolar localization, we constructed plasmids encoding GFP
fused to various regions of CheB: the N-terminal regulatory domain (N), the linker (L) and the C-terminal catalytic
domain (C) (Fig. 1B, see Experimental procedures).
These plasmids were introduced into RP4953 (DcheB)
cells and examined for ability to support chemotaxis. Cells
expressing any fusion did not show chemotaxis, but thoseexpressing the LC fusion spread to some extent (Fig. 2).
The linker might serve to stabilize the CheB structure or
to help the CheB activity, as cells expressing the C fusion
did not spread at all in contrast to those expressing the
LC fusion. We next examined the expression levels ofthese proteins by immunoblotting with anti-GFP antibody(Fig. 3). The C domain fusions were less abundant thanthe N domain fusions. Both the N and the C domain
fusions with the L region expressed at higher levels thanthose without the L region.
We then examined subcellular localization of GFP–CheB derivatives expressed in RP4953 (DcheB) cells(Fig. 5). Whereas the N, L and C fusions did not show any
localization, the NL fusion localized to a cell pole. The LC
fusion also showed significant polar localization, albeit toa lesser extent. When expressed at a higher level (frompSB241), polar localization of the LC fusion was moreevident [Fig. 5, LC (overexp.)]. The N fusion did not localize when expressed from pSB111 but, when overproduced from pSB211, a small fraction of cells wereoccasionally found with faint polar fluorescent spots(Table 1). The L and C fusions did not localize even under
overproducing conditions (expressed from pSB231 and251 respectively; Table 1).
The C-terminal NWETF sequence of the chemoreceptor
is not required for the localization of GFP-CheB
A previous study in this laboratory demonstrated that theMTase CheR localizes to a cell pole by binding to the Cterminal NWETF sequence of a high-abundance MCP(Shiomi et al., 2002). We examined whether the NWETF
sequence is also required for the localization of GFP–CheB. HCB436 cells were transformed first with the plasmid encoding the wild-type or truncated (W550Op, i.e.
lacking the four residues of the NWETF sequence) versionof Tar and then with the H190Y mutant version of GFP–CheB, the NL and LC fusions. All these GFP–CheB fusion
proteins localized to a cell pole even in the absence of the
NWETF sequence (Table 1). This result indicates that the
localization of CheB does not require its interaction with
the NWETF sequence of an MCP.
The P2 domain of CheA serves as a target for the
localization of CheBCheB might interact with the methylation helices of MCPs
or with CheA to localize to a cell pole. The histidine kinase© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

Localization of receptor-modifying enzyme 1055
Fig. 5. Subcellular localization of the GFP
fusion proteins with the various regions of
CheB. Each GFP–CheB fusion protein was
expressed in RP4953 (DCheB) in the presenceof 1 mM arabinose (N, pSB111; NL, pSB121;
L, pSB131; LC, pSB141; C, pSB151) or in
the absence of IPTG or any other inducer[LC (overexp.), pSB241].

CheA consists of the P1 (phosphotransfer), P2 (binding
to CheY and CheB), P3 (dimerization), P4 (kinase) andP5 (coupling to MCPs and CheW) domains (Fig. 6A).
First, strains RP437 (wild type for chemotaxis), RP9535(DcheA) and UU1121 (cheADP2) were transformed withthe plasmid encoding Tar–GFP (Fig. 6B). This Tar–GFP
construct has been shown to localize to cell poles
although its localization pattern may not exactly reflectthat of wild-type Tar (Homma et al., 2004). Immunofluorescence and immunoelectron microscopy show that
MCP clusters occupy part of either or both cell poles(Maddock and Shapiro, 1993), whereas Tar–GFP forms
large polar foci and is always bipolar. Nevertheless, Tar–
GFP is diffused in the absence of CheA and CheW as
observed for wild-type Tar in immunofluorescence andimmunoelectron microscopy (Maddock and Shapiro,
1993) and, therefore, the Tar–GFP fusion can be used to
study essential localization patterns of wild-type Tar inliving cells. Tar–GFP localized to poles of cells expressing
the mutant CheA protein lacking the P2 domain (UU1121),
whereas it localized only partially to poles of cells lacking
CheA (RP9535). Secondly, we transformed the same hoststrains with the plasmid encoding GFP–CheB (Fig. 6B).
Unlike Tar–GFP, GFP–CheB did not localize in UU1121(cheADP2) cells, whereas GFP–CheR localized normallyto a pole (data not shown). These results suggest that theP2 domain of CheA is required for the localization of
CheB. Similarly, the deletion of the P2 domain of CheA
completely abolished polar localization of the NL fusion(Fig. 7). Interestingly, however, the LC fusion localized toTable 1. Polar localization of GFP–CheB and its derivatives.
Host
HCB436 (DMCPs DCheRB)
Plasmid nameProtein fused to GFP

RP4953(DCheB)

pDS900pDS221pDS901pDS902pDS904pDS906
pSB111
pSB121
pSB131
pSB141
pSB151

None
CheR
CheB
CheB-D11K
CheB-D56N
CheB-H190YCheB (N)CheB (NL)
CheB (L)CheB (LC)CheB (C)

–
ND
+
+
+
+
±
+
–
+
–

Tar

Tar-W550Op

No receptor

–
+
+
+
+
+
ND
+
ND
+
ND

–
–
+
+
+
+
ND
+
ND
+
ND

–
–
–
–
–
–
–
–
–
–
–

Wild-type, mutant or truncated versions of GFP–CheB were expressed with 1 mM arabinose alone in RP4953 cells lacking CheB or together
with the full-length or truncated (W550Op) versions of Tar in HCB436 cells lacking all MCPs, CheR and CheB. At least 200 cells were scored ineach observation, which was repeated at least three times. +, > 10%; ±, 0–10%; –, no detectable polar localization; ND, not determined.
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

1056 S. Banno, D. Shiomi, M. Homma and I. Kawagishi
Fig. 6. Effects of cheA deletions on the polar
localization of CheB.
A. The domain organization of CheA (Morrisonand Parkinson, 1997). The domains containing
the phosphorylation site (P1), the CheY/CheBbinding domain (P2), the catalytic domain andthe receptor/CheW-coupling domain are discrete structural and functional domains.
B. Subcellular localization of Tar–GFP (upper,
pDS1020 without an inducer) and GFP–CheB(lower, pDS901 with 1 mM arabinose) in cheA
deletion strains. The fusion proteins wereexpressed in RP437 (Che+), RP9535 (DCheA)
or UU1121 (DP2) cells.

a cell pole in the strain lacking the P2 domain. Even in the
DcheA strain, polar localization of the LC fusion was
detected in a minor population, suggesting that the LC
fusion interacts directly with MCPs that show partial polar
localization in the absence of CheA. The localization patterns of the LC fusions in the DcheA and cheADP2 strains
are reminiscent of those of GFP–CheR. These results
suggest that the catalytic domain does not contribute to
the polar localization of full-length CheB, at least under
the conditions tested, but that the C domain, when liber-

ated, can localize to a pole presumably by interacting with
MCPs, CheW or the CheA domains other than P2.
To confirm that the NL domain of CheB associates with
the P2 domain of CheA, we used a pull-down assay. We
fused glutathione S-transferase (GST) to the P1 and/or
P2 domains of CheA, overexpressed the resulting GST
fusion proteins in RP3098 [D(flhA–D)] cells lacking any
MCP or Che protein and added to glutathione column
chromatography. Crude cytoplasmic fractions of RP3098
cells expressing GFP–CheB, the NL or the LC derivative
Fig. 7. Subcellular localization of GFP-NL(upper, pSB121) and GFP-LC (lower, pSB141)
in cheA deletion strains. The fusion proteins
were expressed in RP437 (Che+), RP9535(DCheA) or UU1121 (DP2) cells in the presenceof 1 mM arabinose.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

Localization of receptor-modifying enzyme 1057
Fig. 8. Binding of CheB to the P2 domain of
CheA, determined by GST pull-down assay.
First, a cytoplasmic fraction containing GST-P1(pSB702) or -P2 (pSB707) and then a cytoplasmic fraction containing GFP–CheB (pSB201),
NL (pSB221) or LC (pSB241) were applied toa glutathione Sepharose 4B column. The column was washed three times, proteins were
eluted twice with glutathione, and all five fractions were applied to SDS-PAGE. GST–CheA
and GFP–CheB were detected by immunoblotting with anti-GST and anti-GFP antibody
respectively. L, cell lysate; F, flowthrough; W,
wash; E, eluate.

were applied to the columns precharged with each GST
fusion protein (Fig. 8). When glutathione was applied,
GFP–CheB was co-eluted with the GST–P1P2 (notshown) or GST–P2 fusion, but not with GST or the GST–
P1 fusion. Similar results were obtained for the NL fusion.
In contrast, the LC fusion was not co-eluted with either
the GST–P1 or the GST–P2 fusion. It is therefore concluded that only the NL domain is responsible for bindingof CheB to the P2 domain of CheA.
Discussion
In this study, we found that the MEase/DAase CheB localizes to a cell pole in an MCP-dependent fashion. The
MTase CheR also localizes to a cell pole in the presenceof MCP (Shiomi et al., 2002). Assembly of such modifying
enzymes around their substrates may enhance the efficiencies of receptor methylation and demethylation/
deamidation. Barkai et al. (2001) and Sourjik and Berg
(2002) suggested that CheB is also involved in signal
amplification. This process might require polar localizationof the protein. The mechanism of polar localization of
CheB was found to differ from that of CheR. Binding to
the P2 domain of CheA recruits CheB to a cell pole,
whereas CheR targets to the C-terminal NWETF
sequence of a high-abundance MCP (Fig. 9). CheB interacts with the NWETF sequence but the affinity is rather
low (Kd ª160 mM) (Barnakov et al., 2001; 2002). CheB
binds to CheA with a Kd value of about 3.2 mM (Li et al.,1995), whereas CheR binds to the NWETF pentapeptide© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

with a Kd value of about 2 mM (Wu et al., 1996), suggesting that this range of affinity may be required and sufficientfor targeting.
Strikingly, the NL and LC domains of the MEase/DAase
CheB, when liberated, localized independently to a cellpole. Binding of the NL domain to the P2 domain of CheA
is responsible for its polar localization, although that is not
the case for the localization of the LC domain. Thus, thelocalization of full-length CheB seems to depend on the
NL domain. It is interesting that the N domain regulates
not only the MEase/DAase activity of the C domain but
also the localization of the protein. Phosphorylation of the
N domain may activate, rather than simply derepress, theC domain as full-length phospho-CheB has a higher
MEase/DAase activity than the isolated C domain (Anandet al., 1998). However, this phenomenon may not bedirectly related to the requirement of the N domain forpolar localization as the in vitro assay did not include CheA.
The linker itself might not be involved in the inhibition of
the MEase/DAase activity (Anand and Stock, 2002), but
its flexibility might help CheB to change its conformationfrom the closed (inactive) form to the open (active) form
(Anand et al., 1998). Phosphorylation of the regulatory
domain is predicted to shift the equilibrium between the
open and closed forms (Anand et al., 1998). The fact that
the NL and LC fusions, but not the N and C fusions, localize
efficiently raises the possibility that the linker might also
be involved in stability and/or localization of the protein.
In wild-type cells, the NL fusion showed very modest
localization, whereas the LC fusion localized nicely

1058 S. Banno, D. Shiomi, M. Homma and I. Kawagishi

Fig. 9. Mechanisms underlying polar localization of the MEase/DAase CheB and the MTase CheR in an E. coli cell. CheB localizes to a cell pole
via the binding of its N-terminal regulatory domain to the P2 domain of the histidine kinase CheA, whereas polar localization of CheR requires
the binding of its b-subdomain to the C-terminal NWETF sequence of the chemoreceptor (MCP). The C-terminal fragment of CheB (consistingof the linker and the C-terminal catalytic domain) also localizes to a cell pole, presumably by interacting with the methylation helices of MCPs,
which might therefore serve as second targets of CheB.

(Fig. 7), a reproducible pattern that seems to be opposite
to that in the cheB mutant cells (Fig. 5). One likely explanation for this difference is that the LC fusion, but not the
NL fusion, competes with wild-type CheB, and so the latterdoes not localize in its presence. In fact, the NL fusion,even when overproduced, did not show a dominantnegative effect on chemotaxis of wild-type cells (S. Banno
and I. Kawagishi, unpublished observation). This interpretation, however, immediately raises further questions,such as why GFP–CheB localizes well in wild-type cells(Fig. 6) and why the LC fusion localizes better in wild-type
cells than in cheB mutant cells. These are difficult to
explain, but one possibility is that the N and C domains
might somehow stabilize the interactions of the C and N
domains with their targets, respectively, either through a

direct N–C interaction or an indirect mechanism, such as
an allosteric effect on the receptor–kinase complex. Theseissues should be addressed experimentally.
Is there any physiological significance of polar localization of the LC fusion? If so, there are two possibilities: (i)a similar fragment might exist in vivo; or (ii) the NL and
LC domains of full-length CheB might bind to different
parts of the receptor–kinase cluster. A C-terminal fragment (ª 21 kDa) of CheB, which results from a proteolytic
cleavage in the linker region, was detected in cell extracts(Simms et al., 1985). We also detected a similar proteolytic fragment of CheB–yellow fluorescent protein(YFP), although the exact cleavage site has not been
determined (data not shown). This proteolytic C fragment
by itself should be able to target to the receptor–kinase© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

Localization of receptor-modifying enzyme 1059
cluster at a cell pole and might serve to maintain a basal
level of MEase/DAase activity or to fine tune signalling,e.g. controlling signal gain. Alternatively, targeting of
CheB to the receptor–kinase cluster could be divided into
two or more distinct stages (e.g. in a hand-over-handmodel discussed below), and the LC domain (as a part offull-length CheB) could be required at a later stage.
In vitro assays demonstrated that CheB is outcompetedby the other response regulator CheY for the binding to
the P1–P2 domains of CheA (Li et al., 1995). Estimatedconcentrations and dissociation constants (Kd) of CheA,
CheY and CheB predict that most CheA molecules in thecell would be free (i.e. no CheY or CheB is bound). Thus,
quick dissociation of CheB from CheA is not necessarily
required. However, CheB might be recycled, because thenumber of CheB molecules is lower than that of MCPs,
CheA and CheY. Indeed, phosphorylation of CheB by
CheA decreases the affinity for the P2 domain, and CheA
inhibits the MEase activity of CheB (Anand and Stock,2002). This would predict that CheB is not always tethered
to the P2 domain of CheA. Rather, the latter domain mightserve only as an initial target of CheB.
Possible second target(s) of CheB would be the methylation helices of an MCP that contain substrate sites ofthe enzyme. This prediction is consistent with the factthat the LC fusion localizes to a pole in the absence of
the P2 domain of CheA. In the case of the MTase CheR,
the two chemotaxis-specific regions bind to the two distinct regions of MCP: the N-terminal domain and theb-subdomain binding to the methylation helices and the
C-terminal NWETF sequence of the chemoreceptor
respectively (Shiomi et al., 2002). Levin et al. (2002) proposed a theoretical model in which CheR moves throughthe receptor cluster in a hand-over-hand fashion with
one of the MCP-binding domains detaching and reattaching to the receptor array before the other domain
dissociates. A similar mechanism might operate for the
CheB function. It is possible that the C domain of CheB
reaches the substrate site on a receptor while the N
domain binds to CheA. The distance from the signallingdomain of MCP to its methylation sites is about 130 Å,which is about twice as long as the linker (20 residues)of CheB even if it is fully extended. However, the P2
domain might lie closer to the methylation sites in the
MCP–CheW–CheA complex. It is also possible that,even after dissociating from the P2 domain, CheB
remains hanging around MCPs, being sequestered in acompartment for the adaptation enzymes as postulatedby Shimizu et al. (2000). In any case, the finding that
CheB and CheR target to the different parts of the polar
receptor–kinase complex indicates that chemotactic
adaptation involves highly ordered arrangement of signalling components.

Experimental procedures
Bacterial strains and plasmids
All strains, plasmids and oligonucleotide primers (for polymerase chain reaction, PCR) used in this study are listed inTables 2, 3 and 4 respectively. The vector plasmid pTrcHisB(Invitrogen) carries the trc promoter, the lacI q gene and thebla gene. The pTrcHisB-based plasmid pDS1020 encodes
the Tar–GFP fusion protein (Homma et al., 2004). The vector
plasmid pBAD24 (Guzman et al., 1995) carries the araBAD
promoter, the araC gene, which encodes the positive andnegative regulator of the araBAD promoter, and the bla gene.
The vector plasmid pACYC184 (Chang and Cohen, 1978)
carries the cat gene. The pACYC184-based plasmid, pLC113(Ames et al., 2002), which carries the wild-type tar coding
region under the control of the nahG promoter, was provided
by J. S. Parkinson. Plasmid pEGFP, which encodes theenhanced GFP, and plasmid pGEX-5X-3, which encodes the
GST, were purchased from Clontech and Amersham Bioscience respectively.
We constructed a plasmid encoding His6-GFP. The 0.7 kb
NheI–EcoRI fragment of pEGFP was subcloned into the vector pTrcHisB to yield pTrc-His6-EGFP. The NcoI–HindIII fragments encoding His6-tagged EGFP from this plasmid were
subcloned into the multicloning site of the vector pBAD24 toyield pDS900.

Site-directed mutagenesis of CheB
Site-directed mutagenesis was performed using a two-step
PCR method essentially as described previously (Umemura
et al., 1998). Plasmid pDS901 encoding wild-type gfp–cheB

Table 2. Bacterial strains used in this study.
StrainGenotype

Reference

HCB436

Dtsr-7021 D(tar-cheB) 2234 Dtrg-100
zbd::Tn5 thr leu his met rpsL136
D(flhD-flhA)4
thi thr leu his met eda rpsL (wild type for chemotaxis)
DcheB m62-16 thi his pyrC46 thyA araD139 Dlac-U169 nalA rpsL
DcheA 1643 thi-1 thr-1 (Am) leuB6 his-4 metF159 (Am)rpsL1356 ara-14 lacY1 mtl-1 xyl-5 tonA31 tsx-78
DcheA 150-247 thi-1 thr-1 (Am) his-4 metF159 (Am)rpsL1356 ara-14 lacY1 mtl-1 xyl-5 tonA31 tsx-78

Wolfe and Berg (1989)

RP3098
RP437
RP4953
RP9535
UU1121© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

Slocum and Parkinson (1983)
Parkinson and Houts (1982)
J. S. Parkinson (personal communication)
Morrison and Parkinson (1994)
J. S. Parkinson (personal communication)

1060 S. Banno, D. Shiomi, M. Homma and I. Kawagishi
Table 3. Plasmids used in this study.
Plasmid

Protein encoded

ParentSourcepACYC184
pBAD24pDS1015pDS1020
pDS900pDS901pDS902pDS904pDS906
pEGFP
pGEX-5X-3
pLC113
pSB111
pSB121
pSB131
pSB141
pSB151
pSB201
pSB211
pSB221
pSB231
pSB241
pSB251
pSB702pSB707
pTrcHisB
pTrcHisC––
Tar-W550Op
Tar–GFP
His6-GFP
His6-GFP–CheB
His6-GFP–CheB-D11K
His6-GFP–CheB-D56N
His6-GFP–CheB-H190Y
GFP
GST
Tar
His6-GFP–CheB (N)His6-GFP–CheB (NL)
His6-GFP–CheB (L)
His6-GFP–CheB (LC)
His6-GFP–CheB (C)His6-GFP–CheB
His6-GFP–CheB (N)His6-GFP–CheB (NL)
His6-GFP–CheB (L)
His6-GFP–CheB (LC)
His6-GFP–CheB (C)GST–CheA (P1)
GST–CheA (P2)
–
–

P15A
pBR322
pLC113
pTrcHisB
pBAD24
pBAD24
pBAD24
pBAD24
pBAD24
pBR322
pBR322pACYC184
pDS900pDS900pDS900pDS900pDS900pTrcHisC
pTrcHisC
pTrcHisC
pTrcHisC
pTrcHisC
pTrcHisC
pGEX-5X-3
pGEX-5X-3
pBR322
pBR322

Chang and Cohen (1978)
Guzman et al. (1995)
Shiomi et al. (2002)
Homma et al. (2004)
This study
This study
This study
This study
This study
Clontech
Amersham Bioscience
Ames et al. (2002)
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
Invitrogen
Invitrogen

was used as a template. The primers (Table 4) were synthesized by Invitrogen. The reaction was carried out using
Pyrobest DNA polymerase (Takara Shuzo) with 25 cycles ofdenaturing at 98∞C for 10 s, annealing at 57∞C for 30 s andextension at 72∞C for 1 min.

Construction of the plasmid encoding the GFP–CheB
fusion protein
The unique HindIII site of the coding region of the wild-typecheB gene was eliminated by introducing a single base sub-Table 4. Primers used in this study.
PrimerPlasmid

Name

Sequencea

pDS900

EGFP-f1(NheI)
EGFP-r1171
CheBf2(BsrGI)
CheBr2(HindIII)
CheB-D11K-f
CheB-D11K-r
CheB-D56N-f
CheB-D56N-r
CheB-H190Y-f
CheB-H190Y-r
CheBN-S134-r
CheBN-L153-r
S134-CheBC-f
L153-CheBC-f
CheA-f(BamHI)
CheA-S167-r(L1)
CheA-R149-f(L1)
CheA-G257-r(L2)

GCGCTAGCATGGTGAGCAAGGGC
CCGCTTACAGACAAGCTGTGACCG
GCTGTACAAGATGAGCAAAATCAGGGCAAGCTTTTAAATACGTATCGCGTTATCTGTCGATAAATCGGCACTGATGCTACAGTGCCGATTTATCGACAGACAACGCTGACGCTGAACGTTGAAATGC
GCATTTCAACGTTCAGCGTCAGCGTTAATTACCCAGTACATGCCGCCC
GGGCGGCATGTACTGGGTAATTAAC
CCCAAGCTTCTAGCTCGCCTTTGCTGC
CCCAAGCTTACAACAACGGCCCCGC
GGGTGTACAAGAGCCTTGCAGCAC
GGGTGTACAAGTTGTTGAGTTCTG
CGGGATCCTGGATATAAGCG
GGCTCGAGGCGACTGACTGC
CGGGATCCGATTAAGTGTGG
GGCTCGAGGCCGGTTGGC

pDS901pDS902bpDS904bpDS906bpSB111cpSB121cpSB131dpSB151epSB702pSB707a. Introduced restriction sites are underlined.
b. CheBf2(BsrGI) and CheBr2(HindIII) were also used.
c. CheBf2(BsrGI) was also used.
d. CheBN-L153-r was also used.
e. CheBr2(HindIII) was also used.
f. Plasmid pSB141 was constructed using S134-CheBC-f and CheBr2(HindIII).
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

Localization of receptor-modifying enzyme 1061
stitution without changing the coding amino acid residue. The
resulting coding region was amplified by PCR using suitableprimers (Table 4) to introduce BsrGI and HindIII sites at their
5¢ and 3¢ ends respectively. The BsrGI–HindIII fragment wascloned between the BsrGI and HindIII sites of the vector
pDS900 to yield the plasmids encoding the wild-type His 6tagged GFP–CheB fusion protein.

Construction of the plasmids encoding GST fusion
proteins with the P1 and/or P2 domain of CheA
The coding regions of the P1 and/or P2 domain of CheA were
amplified by PCR using suitable primers (Table 4) to introduce XhoI and BamHI sites at their 5¢ and 3¢ ends respectively. The PCR products were cloned between the unique
XhoI and BamHI sites of pGEX-5X-3 to yield plasmids encoding GST fused to the P1 and/or P2 domain of CheA.

Detection of GFP–CheB by immunoblotting
RP4953 (DCheB) cells expressing GFP–CheB (full length) or
any of its derivatives (the N, NL, L, LC and C fusions) weregrown at 30∞C for 12 h in TG medium [1% tryptone peptone(Difco), 0.5% NaCl, 0.5% (w/v) glycerol] supplemented with
ampicillin (Ap). The culture was diluted 1:40 into fresh TG
medium supplemented with Ap. Cells were grown with vigorous shaking, harvested at the exponential phase and resuspended in distilled water. The samples were analysed by
SDS-PAGE followed by immunoblotting with anti-GFP antibody (Molecular Probes) or with anti-CheB of Salmonella
typhimurium, which was provided by Dr A. M. Stock.

Observation of subcellular localization of GFP–CheB
Preparation and observation were carried out essentially as
described previously (Shiomi et al., 2002). RP4953 (DCheB)
cells expressing GFP–CheB or its derivatives were grown in
TG medium with Ap. HCB436 (DMCPs DCheRB) cells carrying pLC113 encoding the wild-type Tar (QEQE), its derivative
pDS1015 encoding the truncated version (W550Op) or thevector pACYC184 were further transformed with thepBAD24-based plasmids encoding GFP–CheB or its derivative. The resulting double transformants were grown in TG
medium with Ap and/or Cm supplemented with 1 mM arabinose (for expression of GFP–CheB proteins) and/or 0.5 mM
sodium salicylate (for expression of the Tar proteins). Cells
were grown with vigorous shaking at 30∞C and harvested inlate exponential phase. Cells were washed twice with MLM[10 mM potassium phosphate buffer (pH 7.5), 0.1 mM EDTA,
10 mM DL-lactate, 0.1 mM methionine] and resuspended in
MLM. Small aliquots of the cell suspensions were spotted on
to slide glasses coated with 0.5% agarose and observed
under an inverted fluorescence microscope IX70 (Olympus).
The images were recorded and processed using a cooled
CCD camera CoolNAP-FX/OL (Universal Image Corporation)and the software METAMORPH version 5.0r4 (Roper).

GST pull-down assay for binding of CheB to CheA
A derivative of GST–CheA (P1, P1 + P2 or P2) and a derivative of GFP–CheB (full length, NL or LC) were expressed© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 53, 1051–1063

with 0.5 mM IPTG in the host strain RP3098 [D(flhA–D)]
lacking all Che proteins. The lysate containing each GST–CheA derivative was loaded to a glutathione Sepharose 4B
column (Amersham Biosciences), and then the lysate containing a GFP–CheB derivative was applied to the column.
The column was washed four times, and the proteins were
eluted twice with glutathione. GST–CheA and GFP–CheB
proteins were detected by immunoblotting with anti-GST(Sigma) and anti-GFP antibodies respectively.

Acknowledgements
We thank Drs Michael D. Manson and John S. Parkinson forproviding plasmids and bacterial strains, Dr Ann M. Stock for
antibody, Dr Toshiharu Yakushi for helpful discussion andencouragement, and Dr Howard C. Berg for critical readingof the manuscript. This work was supported in part by grantsin-aid for scientific research from the Japan Society for the
Promotion of Science (to D.S. and I.K.).

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