Microbiology (2002), 148, 3265–3275

Printed in Great Britain

Intragenic suppressors of a mutation in theaspartate chemoreceptor gene that abolishes
binding of the receptor to methyltransferase
Daisuke Shiomi, Michio Homma and Ikuro Kawagishi
Author for correspondence : Ikuro Kawagishi. Tel : j81 52 789 2993. Fax : j81 52 789 3001.
e-mail : i45406a!nucc.cc.nagoya-u.ac.jp

Division of Biological
Science, Graduate School of
Science, Nagoya University,
Chikusa-ku, Nagoya 4648602, Japan

In the chemotaxis of Escherichia coli, receptor methylation is the key processof adaptation. The methyltransferase CheR binds to the carboxy-terminal
NWETF sequence of major chemoreceptors. The substitution of Ala for Trp ofthis sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheRbinding ability. In this study, six independent intragenic suppressors of themutation were isolated. They were divided into two classes. Tar carrying theclass I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showedsignal biases toward tumbling, corresponding to increased activities of thereceptor-associated histidine kinase CheA. These suppressors further reduced
the unstimulated methylation level of Tar-W550A, but allowed slight but
significant stimulation of methylation by aspartate. Some other CheAactivating mutations were also found to serve as class I suppressors. These
results suggest that the class I suppressors compensate for the signal bias of
Tar-W550A caused by its low methylation level and that the NWETF sequence isrequired primarily to maintain an appropriate level of methylation byincreasing the local concentration of CheR around the receptor. The class II
suppressor was a mutation in the termination codon (Op554W) resulting in the
addition of 11 residues containing an xWxxF motif. This revertant Tar
supported chemotaxis and was methylated almost as effectively as wild-type
Tar. This effect was reversed by introducing a mutation in the xWxxF motif.
These results reinforce the importance of the xWxxF motif and suggest thatthe motif does not have to be located at the extreme carboxy terminus.

Keywords : adaptation, chemotaxis, histidine kinase, methylation, revertant

INTRODUCTION

Cell-surface receptors receive extracellular signals (e.g.
by binding ligands) and transduce them into intracellularsignals (e.g. activation or inactivation of kinases). In
many sensory systems, the receptors serve as targets ofregulation (typically via covalent modification) for
adaptation or desensitization, which is essential forhighly sensitive detection of stimuli over a wide range.
Adaptation through covalent modification of receptors
is essential for bacterial chemotaxis (for reviews, see
Manson, 1992 ; Parkinson, 1993 ; Blair, 1995 ; Falke etal., 1997 ; Stock & Surrette, 1996). Escherichia coli and
Salmonella enterica serovar Typhimurium have several
chemoreceptors that are reversibly methylated upon
adaptation. These chemoreceptors are homodimeric
proteins (Milligan & Koshland, 1988), each subunit of0002-5558 # 2002 SGM

which consists of two membrane-spanning regions, aperiplasmic ligand-binding domain and a cytoplasmic
signalling and adaptation domain. In the adaptation
process, four to five glutamate residues (present in the
motif Glu-Glu-X-X-Ala-Ser\Thr, with the underlined
Glu residue being the site of methylation) in two different
helices in the cytoplasmic domain are methylated ordemethylated. These reactions are catalysed by the
methyltransferase CheR and the methylesterase CheB,
respectively. CheR catalyses transfer of a methyl groupfrom S-adenosylmethionine to the side-chain carboxylof glutamate residues (Springer & Koshland, 1977).
The receptor forms a ternary complex with a cytoplasmic histidine kinase (CheA) and an adaptor protein(CheW) (Gegner et al., 1992 ; Schuster et al., 1993 ; Liu etal., 1997). The unliganded (or repellent-bound) receptor
stimulates the activity of CheA, which auto3265

D. Shiomi, M. Homma and I. Kawagishi

phosphorylates itself and then transfers the phosphategroup to the response regulators CheY and CheB (Hesset al., 1988 ; Stock et al., 1988 ; Wylie et al., 1988).
Phospho-CheY binds to FliM, which is a component of
the flagellar motor, and induces the clockwise rotationof the motor, which causes tumbling (Barak & Eisenbach, 1992 ; Welch et al., 1993). Dephosphorylation ofphospho-CheY is facilitated by CheZ. Phospho-CheB
catalyses demethylation of the receptor (Anand et al.,1998 ; Lupas & Stock, 1989). When attractant binds to achemoreceptor, it inhibits the activity of CheA (Borkovich et al., 1989). Consequently, the concentration ofphospho-CheY decreases and the flagella rotate counterclockwise, which results in smooth swimming (Barak &Eisenbach, 1992 ; Welch et al., 1993). The inhibition of
the CheA kinase also decreases the concentration ofphospho-CheB, the active form of the methylesterase,
which reduces demethylation activity and hence increases methylation of the receptors. Methylation of achemoreceptor stimulates CheA to counteract the inhibitory effect of the attractant (Borkovich et al., 1989).
The structure of the cytoplasmic domain of Salmonella
Tar and its relation to kinase regulation were thoroughly
studied by cysteine and disulfide scanning (Bass &Falke, 1998, 1999 ; Bulter & Falke, 1998 ; Danielson etal., 1997). Recently, the three-dimensional structure of acytoplasmic fragment of E. coli Tsr that can activate the
CheA kinase was determined (Kim et al., 1999). The
cytoplasmic fragment (residues 286–526, corresponding
to residues 284–528 of Tar) has a highly α-helicalstructure : each subunit consists of a coiled coil of twolong, antiparallel helices (MH1 and MH2) connected bya U-turn, and the two subunits of a dimer form a quasifour-helix bundle. Methylation is predicted to alter theinteraction between these cytoplasmic helices to switch
signalling states.
A single cell contains thousands of chemoreceptors, but
only hundreds of CheR molecules (Simms et al., 1987).
How can the receptors be effectively methylated? The Cterminal pentapeptide sequence of chemoreceptors iswell conserved among the high-abundance receptors(Tsr, Tar and Tcp), with the consensus being Asn-TrpGlu-Thr(Ser)-Phe. This motif is not conserved in the
low-abundance receptors (Trg and Tap). CheR binds tothis pentapeptide, which is distant from the methylationsites (Wu et al., 1996 ; Djorjevic & Stock, 1998). The
low-abundance receptors have weaker abilities to be
methylated and to allow adaptation when they exist in acell as sole chemoreceptors (Springer et al., 1977 ;Hazelbauer & Engstro$ m, 1980 ; Yamamoto et al.,1990 ; Feng et al., 1997 ; Weerasuriya et al., 1998).
However, when high-abundance and low-abundance
receptors are co-expressed in a cell, the methylationlevel of the latter is enhanced (Kondoh et al., 1979 ;Yamamoto et al., 1990). Furthermore, the addition of
the NWETF sequence to Trg enhances the methylation
and adaptation abilities of that receptor (Feng et al.,1999). Finally, Barnakov et al. (1999, 2001) recently
showed that CheB also binds to the NWETF motif.
Taken together, the pentapeptide sequence seems to
3266

play a crucial role in receptor methylation and adaptation.
Recently, we systematically mutagenized this motif of
Tar and showed that W550 and F553 (especially W550)
play critical roles in the interaction with CheR, probably
through hydrophobic interactions (Shiomi et al., 2000).
Because CheR binding to Tar is almost completely
eliminated by the W550A substitution, the methylationlevel of Tar-W550A is lower than that of wild-type Tarboth in the absence and the presence of aspartate (Shiomi
et al., 2000). However, methylation of Tar-W550A is
still slightly enhanced by aspartate (Shiomi et al., 2000).
The methylation level of Trg, which does not have a
CheR-binding motif, is also regulated to some extent by
its specific attractant ribose or galactose (Feng et al.,1999). To study the physiological role of the NWETF
motif further, we isolated intragenic suppressors of theW550A mutation. Six suppressors were isolated andwere divided into two classes : five class I suppressors,which generated a compensatory tumbling bias (CheA
kinase-activating) and one class II suppressor that
restored the affinity of Tar for CheR.
METHODS
Bacterial strains and plasmids. All E. coli K-12 strains used in
the chemotaxis assay lack all four MCPs (i.e. Tsr, Tar, Trg
and Tap) though they retain the related redox sensor Aer.
Strain HCB339 [∆(tar-tap)5021 ∆tsr-7028 trg : : Tn10 thr leu his
met rpsL136] (Wolfe et al., 1987) has all of the Che proteins
involved in general chemotaxis (i.e. CheA, CheW, CheY,
CheZ, CheB and CheR). Strain HCB436 [∆tsr-7021 ∆(tarcheB)2234 ∆trg-100 zbd : : Tn5 thr leu his met rpsL136] (Wolfe& Berg, 1989) lacks CheB and CheR, and strain HCB1262[∆(cheA-cheZ) : : zeoR thr leu his met rpsL136] (Shiomi et al.,2000) lacks all the Che proteins.

Plasmids used in this work are listed in Table 1. The pBR322based plasmid pNI101 (Nishiyama et al., 1997) carries thewild-type tar gene. Plasmid pOKU121 (Shiomi et al., 2000)
derives from pNI101 and encodes Tar-W550A. Plasmid
pNT201 (Borkovich et al., 1989) carries the wild-type tar gene
under the control of the tac promoter and was used tooverexpress the Tar protein. Plasmid pKB23 (Borkovich et al.,1992) is based on pACYC184 and carries the wild-type cheR
gene placed downstream of the tac promoter.
Construction of plasmids expressing mutant Tar proteins.

Plasmids carrying class I suppressor tar genes without theoriginal W550A mutation were constructed as follows. The
0n8 kb XbaI–MluI fragment from pDS303 (W550A-G278A) or
pDS305 (W550A-G278C) was cloned into the corresponding
region of pNI101 to yield pDS303W (G278A) and pDS305W(G278C), respectively. The 0n4 kb EcoT22I–PvuII fragmentfrom pDS301 (W550A-G278A-L488M) was cloned into thecorresponding region of pNI101 and pOKU121 to yield
pDS311 (L488M) and pDS321 (W550A-L488M), respectively.
The 0n5 kb EcoRV–EcoT22I fragments containing the W192R,
A198E, V201E, V202L, H267D or T303I mutations werecloned into the corresponding region of pNI101 or pOKU121
to yield pDS390W, pDS390A, pDS391W, pDS391A,
pDS392W, pDS392A, pDS393W, pDS393A, pDS394W,
pDS394A, pDS395W or pDS395A, respectively.
Plasmid pNI101 encoding wild-type Tar has two NdeI sites :Binding of the chemoreceptor to methyltransferase
Table 1. Tar-encoding plasmids used in this study
Plasmid

Encoded Tar protein

Parent plasmid

pDS101pDS103pDS301pDS302pDS303pDS303W
pDS304pDS3042
pDS3043
pDS305pDS305W
pDS306
pDS311
pDS321
pDS374pDS390W
pDS390A
pDS391W
pDS391A
pDS392W
pDS392A
pDS393W
pDS393A
pDS394W
pDS394A
pDS395W
pDS395A
pDS396W
pDS396A
pDS397W
pDS397A
pDS398W
pDS398A
pDS399W
pDS399A
pNI101
pNI101N
pNT201
pOKU121
pOKU151

Wild-type
W550A
G278A-L488M-W550A(original suppressor)
T334A-W550A (original suppressor)
G278A-W550A (original suppressor)
G278A
W550A-Op554W (original suppressor)
W550A-Op554A
F553A-Op554W
G278C-W550A (original suppressor)
G278C
A398T-W550A (original suppressor)
L488M
L488M-W550A
W550A-Op554W
W192R
W192R-W550A
A198E
A198E-W550A
V201E
V201E-W550A
V202L
V202L-W550A
H267D
H267D-W550A
T303I
T303I-W550A
G278I
G278I-W550A
G278R
G278R-W550A
G278S
G278S-W550A
G278T
G278T-W550A
Wild-typeWild-typeWild-type
W550A
F553A

pNT201pDS101
pOKU121
pOKU121
pOKU121
pNI101
pOKU121
pOKU121
pOKU151
pOKU121
pNI101
pOKU121
pNI101
pOKU121
pDS101
pNI101
pOKU121
pNI101
pOKU121
pNI101
pOKU121
pNI101
pOKU121
pNI101
pOKU121
pNI101
pOKU121
pNI101N
pOKU121
pNI101N
pOKU121
pNI101N
pOKU121
pNI101N
pOKU121
pAK101
pNI101
pBR322
pNI101
pNI101

one is in the tar gene and the other is in the sequence derived
from pBR322. The latter site was eliminated by blunt-ending
and ligation to yield pNI101N. Site-directed mutagenesis ofresidue G278 was carried out by a two-step PCR method(Landt et al., 1990). The resulting 0n3 kb NdeI–EcoT22I
fragments containing the G278I, G278R, G278S or G278T
substitution were cloned between NdeI and EcoT22I sites of
pNI101N to yield pDS396W, pDS397W, pDS398W or
pDS399W, respectively. The 0n5 kb EcoRV–EcoT22I fragments from pDS396W, pDS397W, pDS398W or pDS399W
were cloned into the corresponding region of pOKU121 toyield pDS396A, pDS397A, pDS398A or pDS399A, respectively.
Plasmids overproducing Tar-W550A or W550A-Op554W

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Nishiyama et al. (1997)
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Borkovich et al. (1989)
Shiomi et al. (2000)
Shiomi et al. (2000)

were constructed as follows. The parental plasmid pNT201,
which has two AvaI sites, was partially digested with AvaI,
blunt-ended and ligated to yield pDS101. The 1n4 kb XbaI–
AvaI fragments from pOKU121 (W550A) or pDS304 (W550AOp554W) were cloned into the corresponding region of
pDS101 to yield pDS103 (W550A) and pDS374 (W550AOp554W), respectively.
Swarm assay for chemotaxis. Swarm assays were performed

with tryptone semisolid agar (1 % tryptone, 0n5 % NaCl, 0n3 %agar) or minimal semisolid agar [10 mM potassium phosphatebuffer (pH 7n0), 1 mM (NH ) SO , 1 mM MgSO , 1 mM%# %%glycerol, 1 µg thiamine ml−", 0n1 mM threonine, 0n1 mM
leucine, 0n1 mM histidine, 0n1 mM methionine, 0n3 % agar]
supplemented with various concentrations of aspartate. When
3267

D. Shiomi, M. Homma and I. Kawagishi
necessary, 50 µg ampicillin ml−" and\or 25 µg chloramphenicolml−" was added. Cell suspensions (2 µl each, about 4i10'
cells) were spotted onto swarm plates, which were thenincubated at 30 mC for 10–20 h.
Temporal assay of chemotactic response. Temporal stimulation assays for chemotaxis were carried out as described
previously (Okumura et al., 1998). Cells were grown at 30 mC
in TG medium [1 % tryptone, 0n5 % NaCl, 0n5 % (w\v)
glycerol] supplemented, when necessary, with 50 µg ampicillin ml−" and\or 25 µg chloramphenicol ml−". Cells were
harvested in late-exponential phase, washed with MLM
medium [10 mM potassium phosphate buffer (pH 7n0),0n1 mM EDTA, 10 mM -lactate, 0n1 mM methionine],
resuspended in MLM medium and incubated at room temperature for 30 min. For dose–response assays, the swimming
patterns of the cells were observed with a dark-field microscope immediately after the addition of chemoeffector. For
adaptation assays, a chemoeffector was added to the cellsuspension, and small aliquots were taken at intervals for
microscopic observation. The smooth-swimming fraction of
the cells was measured using an Argus-10 image processor(Hamamatsu Photonics).
Analysis of receptor methylation by immunoblotting. Taking
advantage of the fact that methylation of a receptor increases
its mobility in SDS-PAGE (Boyd & Simon, 1980 ; Chelsky &Dahlquist, 1980 ; DeFranco & Koshland, 1980 ; Engstro$ m andHazelbauer, 1980), receptor methylation was examined withimmunoblotting. HCB339 (∆MCPs) cells expressing the revertant Tar proteins were grown at 30 mC for 12 h in TG
medium [1 % tryptone, 0n5 % NaCl, 0n5 % (w\v) glycerol]
supplemented with ampicillin. The culture was diluted 1 : 40
into fresh TG medium supplemented with ampicillin. Cells
were grown with vigorous shaking, harvested in the lateexponential phase and resuspended in 100 µl distilled water.
The samples were analysed by SDS-PAGE followed byimmunoblotting with anti-Tsr (Iwama et al., 1997), whichcross-reacts with Tar.
Analysis of receptor methylation by radiolabelling. Methylation of Tar was assayed by radiolabelling, as described
previously (Nishiyama et al., 1997). Cells in medium consistingof 10 mM potassium phosphate buffer (pH 7n0), 0n1 mM
EDTA, 10 mM -lactate, 3 µM methionine and 0n2 mg ml−"chloramphenicol were incubated at 30 mC with vigorous
shaking. After 30 min, 3n3 µM [methyl-$H] methionine[8n5 Ci mmol−" (314n5 GBq mmol−") ; Amersham] was added
to the cells and this suspension was shaken for another 30 min.
The cell suspension was divided into two aliquots, which wereshaken for 15 min in the presence and absence of 10 mM
aspartate, respectively. The reaction was terminated by adding
trichloroacetic acid, and methyl-labelled proteins wereanalysed by SDS-PAGE followed by fluorography.

RESULTS
Isolation of intragenic suppressors of the W550A
mutation

When a plasmid encoding Tar-W550A is introduced
into HCB339 (∆MCP) cells, which lack all of the methylaccepting chemoreceptors except Aer, the resulting
transformant cells respond to aspartate but do not adapt
and hence cannot swarm in tryptone semisolid agar
(Shiomi et al., 2000). Starting with this strain, we isolated
60 independent spontaneous pseudorevertants that3268.................................................................................................................................................

Fig. 1. Intragenic suppressors of the W550A mutation of tar.
Among 60 independent swarm-positive revertants from HCB339(∆MCP) cells carrying plasmid pOKU121 (encoding Tar-W550A),
six proved to have mutations in the tar gene. They retained theoriginal mutation and had the following second-site suppressor
mutations : G278A, G278A-L488M, G278C, T334A, A398T and
Op554W. The mutation sites are indicated with stars in the tar
coding region with the original (with residue numbers) and themutant amino acid residues (the corresponding nucleotide
sequences are shown in parentheses). The double mutations(G278A-L488M) are marked with open boxes. Op554 indicates
the termination codon (TGA), the mutation of which results inthe addition of the 11 residues (WRVFTNAVIAA) to the C
terminus of Tar-W550A. Open circles indicate methylation sites.
TM1 and TM2, the first and the second transmembrane
helices ; MH1 and MH2, the first and the second methylation
helices.

spread in tryptone semisolid agar. Plasmid DNA prepared from these revertants was reintroduced into thehost strain, and plasmids from six independent pseudorevertants gave swarm-positive transformants. The
nucleotide sequences of the tar genes from the six
revertants were determined. All of them retained theoriginal W550A mutation and had second-site suppressors (Fig. 1) : four of them were single missense
mutations (G278A, G278C, T334A and A398T), one
had two mutations causing two amino acid changes(G278A-L448M), and one had a mutation in thetermination codon (Op554W : TGA to TGG) that
resulted in the addition of 11 residues at the C terminusof Tar.
Swarming abilities of cells expressing the revertant
Tar receptors

HCB339 cells expressing these revertant Tar receptors
were tested for their swarming abilities in semisolid agar
plates (Fig. 2). In tryptone semisolid agar, cells expressing the Op554W receptor formed a slightly larger
swarm than cells expressing wild-type Tar. The cells
expressing the other revertant Tar proteins formed
smaller swarms than cells expressing wild-type Tar [Fig.
2A, B(a)]. Thus, the suppressors can be divided into twoclasses : five class I suppressors (G278A-L488M, T334A,
G278A, G278C and A398T), which restore swarming
only partially, and one class II suppressor (Op554W),
which restores full swarming ability. Essentially similarBinding of the chemoreceptor to methyltransferase.....................................................................................................

Fig. 2. Swarming ability of HCB339 cells
expressing the revertant Tar receptors.
Aliquots (2 µl each) from three independent
fresh overnight cultures were spotted onto
ampicillin-supplemented tryptone [A, B(a)]or minimal semisolid agar containing 10−4 M[B(b)] or 10−5 M [B(c)] aspartate and theplate was then incubated at 30 mC. The
swarm diameter of cells carrying the vector
pBR322 (none) was subtracted from thoseof cells expressing each receptor to obtainnet swarm diameters. The relative swarm
diameter is defined as the net swarm
diameter normalized to the mean diameterof swarms formed by cells expressing wildtype Tar (B). The relative swarm diameters in
the presence of 10−5 M aspartate [B(c)] werenormalized to the mean diameter of swarms
formed by cells expressing wild-type Tar in
the presence of 10−4 M aspartate [B(b)].

results were obtained when minimal semisolid agar
containing 10−% M aspartate was used [Fig. 2B(b)]. In
contrast, in minimal semisolid agar containing 10−& Maspartate, cells expressing Tar-W550A formed a larger
swarm than cells expressing any class I revertant Tar,
though the swarm was much smaller than that of cells
expressing wild-type Tar [Fig. 2B(c)]. It is possible thatthe gradient formed in agar containing 10−& M aspartate
are not steep enough to generate smooth swimming withthe class I revertants that cause tumbling bias (seebelow). On the other hand, the gradient formed with
10−% M aspartate may be steep enough to stimulate theclass I revertants but may not allow cells expressing
Tar-W550A to adapt.
Adaptation abilities of cells expressing the revertant
Tar receptors

Swarming is influenced not only by the ability of cells to
respond to and to adapt to attractants but also by cell
growth and signal (smooth-swimming versus tumbling)bias. We therefore examined swimming bias and chemo-

tactic responses of HCB339 cells expressing each revertant Tar in the temporal stimulation assay. The basal
swimming bias of cells expressing Tar-W550A was
similar to that of cells expressing wild-type Tar (Fig.
3A). However, they did not adapt to aspartate (Fig. 3A)even after 13 min (data not shown). We determined
minimal aspartate concentrations that elicit 100 %attractant responses mediated by the revertant proteins(data not shown). These concentrations were used in thefollowing adaptation assays. Cells expressing any class I
revertant Tar were tumbly (Fig. 3B). However, they
became smooth-swimming immediately after additionof 10−& or 10−% M aspartate (i.e. attractant responses)
and they gradually adapted to return their original
tumbly behaviours (Fig. 3B). In contrast, cells expressing
the class II revertant Tar (W550A-Op554W) behaved
similarly to cells expressing wild-type Tar both in termsof their basal swimming bias and adaptation (Fig. 3B).
These results suggest that the class I suppressors bias thesignal output toward tumbling (i.e. CheA-activating
mutations) whereas the class II suppressor restore theaffinity of Tar for CheR.
3269

D. Shiomi, M. Homma and I. Kawagishi

is modulated in response to aspartate. We therefore
examined the methylation patterns of the revertant Tar
proteins (Fig. 4). Although the expression level of theclass II revertant Tar receptor was lower than that ofwild-type Tar (Fig. 4A), the unstimulated (k) andaspartate-stimulated (j) methylation levels of TarW550A-Op554W were similar to those of wild-type Tar(Fig. 4), supporting the notion that the class II revertant
restores the affinity for CheR. In contrast, the methylation levels of the class I revertant Tar receptors wereeven lower than that of the original mutant Tar-W550A
under both the unstimulated (k) and the aspartatestimulated (j) conditions (Fig. 4B). This result suggeststhat the class I revertants do not restore affinity for
CheR. However, these revertants still showed slightly
but significantly enhanced methylation in the presenceof aspartate, a property consistent with their ability toadapt.
Analysis of the class I suppressors

To characterize the class I suppressor mutations further,
we constructed Tar receptors carrying G278A or G278C
without the original W550A substitution. HCB339 cells
expressing Tar-G278A or G278C were then tested forchemotactic ability (Fig. 5A). These cells formed larger
swarms than cells expressing the W550A receptor. Cells
expressing Tar-G278A swarmed slightly faster than cells
expressing Tar-G278C and as fast as cells expressing
wild-type Tar. These cells responded and adapted toaspartate as well as cells expressing the corresponding
receptor with W550A (data not shown). Immunoblotting with anti-receptor serum showed that the
methylation patterns of the G278A and G278C receptors
were similar to those of the corresponding receptors
with W550A (data not shown). These results suggestthat G278A and G278C are tumbling-bias mutations as
Trammell & Falke (1999) showed for Salmonella Tar. It
is likely that T334A and A398T also cause a tumblingbias (see below).
.................................................................................................................................................

Fig. 3. Adaptation ability of HCB339 cells expressing TarW550A or its revertant derivatives. (A) Time-courses of
aspartate responses of HCB339 cells expressing the wild-type or
W550A receptor. At the time indicated by an arrow, 10−5 M
aspartate was added. (B) Time-courses of aspartate responses of
HCB339 cells expressing each revertant receptor. Aspartate wasadded at 10−5 M (G278A-L488M, G278A and Op554W) or 10−4 M(T334A, G278C and A398T).

Methylation patterns of the revertant Tar receptors

Methylation of chemoreceptors is the key process ofadaptation. HCB339 (∆MCP) cells expressing any of therevertant Tar proteins adapted to aspartate (Fig. 3B),
whereas HCB436 cells, which lacks CheB and CheR aswell as all four chemoreceptors (∆MCP ∆CheB ∆CheR),
did not adapt to aspartate when they expressed any of
the revertant receptor proteins (data not shown), implying that methylation of all the revertant Tar proteins
3270

Next, to examine the nature of the L488M mutation, weconstructed plasmids encoding Tar carrying the L488M
mutation with or without the W550A mutation.
HCB339 cells expressing Tar-L488M swarmed as fast as
cells expressing wild-type Tar, whereas cells expressing
the corresponding receptor with W550A failed to swarm(Fig. 5A). This result indicates that the L488M mutation
does not suppress the W550A mutation and was just asilent second change in the G278A-L488M receptor.
Effects of kinase-activating mutations on the W550A
receptor

The results described above raised the possibility that
other tumbling-bias (kinase-activating) mutations may
also serve as class I suppressors of the W550A substitution. We therefore introduced several known
tumbling-bias mutations into the W550A receptor. First,
we focused on residue G278 in the first methylation helix
because this residue is a critical site of kinase regulation

Binding of the chemoreceptor to methyltransferase.....................................................................................................

Fig. 4. Methylation levels of the revertant
Tar receptors. Methylation of the wild-type,
W550A, and revertant receptors was assayedby immunoblotting with anti-Tsr (A) andradiolabelling (B), as described in Methods,
in the absence (k) or presence (j) of 10
mM aspartate. For each sample, the intensity of the receptor band on fluorographs
was divided by that of methylatable EF-Tu
protein, which serves as an internal control.
The resulting value was normalized to thatof wild-type Tar in the absence of aspartate
to obtain the relative level of methylation.

(Trammell & Falke, 1999). The residue was replaced bySer, Thr, Arg or Ile. Cells expressing Tar-G278T-W550Aor Tar-G278R-W550A formed a similar swarm to cells
expressing the class I revertant Tar receptors, whereas
cells expressing Tar-G278S-W550A formed a smallerswarm (Fig. 5B). Cells expressing Tar-G278I-W550A
failed to swarm (Fig. 5B), which is consistent with thefact that the Tar-G278I mutant protein cannot regulate
kinase activity in response to aspartate (i.e. it constitutively activates CheA kinase) (Trammell & Falke,1999).
Next, we examined the mutations that were initially
isolated as suppressors of the A19K substitution thatabolishes the signalling ability of Tar without impairing
the aspartate-binding ability (Oosawa & Simon, 1986).
All of them are single-residue substitutions in the second
transmembrane helix (TM2 ; W192R, A198E, V201E
and V202L) or in the first methylation helix (MH1 ;H267D and T303I) that cause a wide range of tumbling
biases (Tatsuno et al., 1996 ; Umemura et al., 1998). In
the absence of chemoeffector, cells expressing TarA198E-W550A or Tar-T303I-W550A tumbled incessantly, whereas cells expressing the other mutant receptors showed various degrees of signal bias (Fig. 5C).
In swarm assay, cells expressing Tar-A198E-W550A or
Tar-T303I-W550A formed a similar swarm to cells
expressing the class I revertant receptor, whereas cells
expressing any of the other constructs failed to swarm(Fig. 5D). Thus, the A198E (in TM2) and T303I (inMH1) substitutions, but not the other substitutions
which caused weaker tumbling biases, can serve as
suppressors of the W550A substitutions. Taken together, a strong (but not constitutive) tumbling bias
seems to be required (and probably sufficient) forsuppression of the W550A substitution.
Analysis of the class II suppressor

The results described above suggest that the class II
suppressor restores the affinity of Tar-W550A for CheR.

We tried to detect binding of CheR to inverted membrane vesicles containing Tar-W550A-Op554W. However, the level of binding was much lower than that tomembranes containing wild-type Tar (data not shown).
The class II revertant Tar receptor has 11 additional
residues (WRVFTNAVIAA) due to the mutation of thetermination codon (Op554W). This carboxy-terminal
extension contains an xWxxF motif that is especially
important for the interaction with CheR (Djordjevic &Stock, 1998 ; Shiomi et al., 2000) and therefore mightserve as a (weak) binding site for CheR. To examine thispossibility, we first examined the effect of the Op554W
mutation on the Tar receptor with the F553A mutation,
which is less severe than W550A (Shiomi et al., 2000),
probably because the phenylalanine residue is lesscritically involved in the interaction with CheR than thetryptophan residue (Djordjevic & Stock, 1998). Cells
expressing Tar-F553A-Op554W swarmed as fast as cells
expressing Tar-W550A-Op554W (Fig. 6), indicatingthat the Op554W mutation can also suppress the defectcaused by the F553A mutation. We next examined theeffect of Op554A substitution (i.e. the loss of the xWxxF
motif in the 11 additional sequences) on the W550A
receptor. Cells expressing Tar-W550A-Op554A failed
to swarm, suggesting that the tryptophan residue at
position 554 is critical for suppression. These results are
consistent with the interpretation that the class II
suppressor partially restores binding of CheR to thereceptor by creating an xWxxF motif in the carboxyterminal additional sequence.
DISCUSSION

In this study, we isolated six independent intragenic
suppressors from the mutant tar gene encoding theW550A residue substitution. These suppressors could be
divided into two classes. When expressed as the solechemoreceptors in a cell, the five class I revertant
receptors supported chemotaxis less effectively thanwild-type Tar. Their methylation levels were even lower
3271

D. Shiomi, M. Homma and I. Kawagishi.....................................................................................................

Fig. 5. Characterization of class I
suppressors. (A) Swarming ability of HCB339
cells expressing the wild-type, W550AG278A-L488M,
W550A-G278A,
W550AG278C, G278A, G278C, L488M or no receptor(None). (B) Swarming ability of HCB339 cells
expressing the W550A receptor carrying an
additional tumbling-bias mutation (G278A,
G278C, G278I, G278R, G278S, or G278T) as
well as those expressing the wild-type, or noreceptor (None). (C) Swimming bias of
HCB339 cells expressing the W550A receptor
with or without (–) an additional A19Ksuppressor mutation (A198E, V201E, V202L,
H267D, or T303I) as well as those expressing
the wild-type receptor. (D) Swarming abilityof HCB339 cells expressing the W550A
receptor with or without an additional
A19K-suppressor mutation (A198E, V201E,
V202L, H267D, or T303I) as well as thoseexpressing the wild-type, or no receptor(None). Swarming was tested in tryptone
semisolid agar containing ampicillin. The
relative swarm size is defined as in thelegend to Fig. 2.

than that of Tar-W550A, but methylation levels increased significantly upon the addition of aspartate. The
class II revertant receptor, in which the original termination codon was altered to a UGG (Trp) codon led
to the addition of 11 additional residues, supported
chemotaxis, and was methylated almost as effectively as
wild-type Tar.
Of the class I suppressors, G278A and G278C had
already been characterized by Trammell & Falke (1999) :the receptors with these mutations superactivate CheA
autophosphorylation in the absence of aspartate, but inthe presence of aspartate the mutant receptors fully andpartially inhibit the kinase, respectively. The receptors
with the other class I suppressors (T334A and A398T)
also exhibited tumbling-biased signalling and their
methylation levels were lower than that of Tar-W550A,
suggesting that these mutations also render the signal
bias toward the activation of CheA. We also showedthat some other tumbling-bias mutations, which allow
regulation of the activity of CheA kinase in response to3272

aspartate, are also able to suppress the W550A suppression regardless of the place of the second site
mutation. However, among the tumbling-bias mutations tested, those which cause weaker tumbling biases
or which cause constitutive activation of CheA failed tosuppress the W550A mutation. Therefore, we conclude
that the class I suppressors compensate for an excessively
smooth-swimming (i.e. CheA-inactivating) bias caused
by decreased methylation of Tar-W550A.
Wild-type Tar inhibits CheA activity in the presence of
aspartate, but increased methylation of Tar during
adaptation to aspartate restores the CheA- stimulating
activity. Methylation of Tar-W550A can also be stimulated to a certain extent by aspartate, but this enhancement of methylation may be too slight to reactivate
CheA in the presence of aspartate. The class I revertant
Tar receptors can inhibit the CheA activity in response
to aspartate, but they still can stimulate CheA when
their methylation is enhanced by aspartate (i.e. when thecell adapts to aspartate) although their absolute levels of

Binding of the chemoreceptor to methyltransferase

foreign residues (WRVFTNAVIAA) due to the mutationof the termination codon (Op554W). The results presented here suggest that the class II suppressor partiallyrestores binding of CheR to the receptor by creating an
xWxxF motif in the carboxy-terminal additional sequence. This means that the CheR-binding sequence
may not have to be located at the extreme carboxy
terminus of the chemoreceptor.

.................................................................................................................................................

Fig. 6. Swarming ability of HCB339 cells expressing the W550A
or F553 receptor with or without (k) an additional mutation
Op554W or Op554A as well as those expressing the wild-type,or no receptor (None). Swarming was tested in tryptone
semisolid agar containing ampicillin. The relative swarm size isdefined as in the legend to Fig. 2.

methylation are lower than that of wild-type Tar,
presumably because of ‘constitutive’ activation of CheA,
and hence the methylesterase CheB. This difference in
CheA activation between the methylated and unmethylated ligand-occupied revertant receptors may beenough to support chemotaxis to some extent.
The mechanism by which the class I mutations suppress
the defect introduced by the W550A substitution demonstrates the robustness of the chemotaxis system (Barkai& Leibler, 1997 ; Alon et al., 1999). Alon et al. (1999)
showed that chemotaxis is largely independent of thebasal swimming bias and the adaptation time. Although
the revertants failed to support swarming in minimal
semisolid agar containing 10−& M aspartate, the requirement for higher concentrations of aspartate isreadily explained by the suppression mechanism described above. The tumbling-biased signals produced by
the class I receptors may be too strong to be suppressedby 10−& M aspartate.
Presumably, the class II revertant receptor partiallyrestores binding of CheR to the receptor. When expressed as a sole chemoreceptor in a cell, this receptor
supports chemotaxis and a wild-type signal bias, and it
is methylated almost as effectively as wild-type Tar. We
examined binding of CheR to inverted membrane
vesicles containing the class II receptor, but the level ofbinding was much lower than to membranes containing
wild-type Tar (data not shown), suggesting that theaffinity of the revertant protein for CheR is weaker thanthat of wild-type Tar. This revertant Tar receptor has 11

The current understanding of the regulation of methylation of chemoreceptors is as follows. Binding of an
attractant to a chemoreceptor leads to inhibition of
CheA (Borkovich et al., 1989), and hence decreases
phosphorylation of CheB (Hess et al., 1988 ; Lupas &Stock, 1989) to decrease the rate of demethylation. The
attractant-bound receptor becomes a better substrate
for CheR and a poorer substrate for CheB, resulting in a
net increase in methylation of the liganded receptor(Springer et al., 1977 ; Wang & Koshland, 1980). Since
the methylation levels of the class I revertant Tar
proteins were lower than the methylation level of TarW550A, we conclude that the affinity of Tar for CheR
was not restored by the class I suppressors. However,
the methylation levels of these receptors were slightly
but significantly enhanced by aspartate, suggesting thatbinding of CheR to the NWETF sequence is not strictly
required for the regulation of the methylation level ofthe chemoreceptor.
What is the role of the pentapeptide sequence then? It is
likely that the pentapeptide motif is required formaintaining an appropriate level of receptor methylation by increasing the local concentration of CheR
around the receptor cluster in the cell. This interpretation is consistent with the following facts : (i) CheR ismuch less abundant than the receptor molecules (Simms
et al., 1987) ; (ii) the overproduction of CheR suppresses
the defect of the mutations in the pentapeptide sequence(Okumura et al., 1998 ; Shiomi et al., 2000) ; (iii) thereceptor molecules form a cluster with CheW and CheA
at a cell pole (Maddock & Shapiro, 1993 ; Lybarger &Maddock, 2000) ; (iv) a receptor dimer with the CheRbinding sequence can help methylation of a neighbouring receptor dimer without the CheR-binding
sequence (Le Moual & Koshland, 1997 ; Li et al., 1997) ;(v) GFP-CheR localized to cell poles in the presence, but
not in the absence, of a chemoreceptor with the NWETF
sequence (Shiomi et al., 2002).
Okumura et al. (1998) showed that cells expressing
mutant Tcp (NWESLA) respond to, but do not adapt to
the repellent glycerol. Barnakov et al. (1999, 2001)
demonstrated that CheB also binds to the NWETF
peptide. We also found that the W550A receptor serves
as a poorer substrate of CheB (unpublished results).
These findings suggest that the NWETF sequence serves
also as a modulator of CheB activity. The intragenic
suppressors of the W550A mutation isolated in thisstudy are either CheA-activating mutations or a mutation that seemingly partially restores the affinity of
Tar for CheR. Moreover, the same original mutation
can be suppressed by the overproduction of CheR
3273

D. Shiomi, M. Homma and I. Kawagishi(Shiomi et al., 2000). Therefore, at least under theconditions tested, binding of the receptor to CheR may
be more important (or rate-limiting) to support chemotaxis than that to CheB. It is also possible that theW550A mutation has a more severe effect on binding of
CheR than that of CheB. Further analyses will be
required to address these issues.
ACKNOWLEDGEMENTS
We thank Hisashi Okumura for examining conditions ofrevertant screening, Dr Tohru Umemura, Kenji Fukami and
Tatsuya Furihata for helping with plasmid construction andnucleotide sequencing, and Dr Ken Sato for helpful discussion
and technical advice. We thank Dr Michael D. Manson (Texas
A & M University) for critically reading the manuscript. This
work was supported in part by grants-in-aid for scientificresearch from the Society for the Promotion of Science (toD. S. and I. K) and from the Takeda Science Foundation(to I. K.).

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.................................................................................................................................................

Received 28 February 2002 ; revised 4 June 2002 ; accepted 21 June 2002.

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