JOURNAL OF BACTERIOLOGY, Mar. 2000, p. 1437–1441
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Vol. 182, No. 5

Mutational Analysis of Ligand Recognition by Tcp, the Citrate
Chemoreceptor of Salmonella enterica Serovar Typhimurium
TOMONORI IWAMA,1 KO-ICHIRO NAKAO,1 HIROSHI NAKAZATO,2† SHUZO YAMAGATA,1
MICHIO HOMMA,2 AND IKURO KAWAGISHI2*
Department of Biotechnology, Division of Utilization of Biological Resources, Faculty of Agriculture,
Gifu University, Gifu 501-1193,1 and Division of Biological Science, Graduate School of
Science, Nagoya University, Chikusa-ku, Nagoya 464-8602,2 Japan
Received 8 September 1999/Accepted 8 December 1999

The chemoreceptor Tcp mediates taxis to citrate. To identify citrate-binding residues, we substituted cysteine
for seven basic or polar residues that are chosen based on the comparison of Tcp with the well-characterizedchemoreceptors. The results suggest that Arg-63, Arg-68, Arg-72, Lys-75, and Tyr-150 (and probably otherunidentified residues) are involved in the recognition of citrate.
The closely related enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium have multiple transmembrane receptors that mediate chemotactic responses toamino acids, non-PTS sugars, and other attractants and repellents (2, 11, 25, 26, 34, 35). Some receptors (Tar for aspartate,
Tsr for serine, and Trg for ribose and galactose) are found inboth species. Others are species specific. Belonging to thelatter class is the Salmonella serovar Typhimurium-specificchemoreceptor Tcp, which mediates taxis to citrate and a divalent cation-citrate complex and away from phenol (39). Salmonella serovar Typhimurium, but not E. coli, can utilize citrate
as a sole carbon source.
Ligand recognition by Tar and Tsr has been studied extensively using mutagenesis (13, 21, 22, 30, 38), chemical modification (14, 15, 17), X-ray crystallography (28, 40), and computer simulation (19). Mutations at the aspartate-binding
residues of Tar cause defects in the aspartate-sensing ability
without affecting the repellent-sensing ability or other receptor
functions (13, 21, 30, 38), although in some cases the maltosesensing ability is also affected due to a partial overlap of thebinding sites for aspartate and the complex of maltose andmaltose-binding protein (13). Tcp is homologous to Tar andTsr, but it recognizes the non-amino acid ligand citrate. Identification of the citrate-binding residues of Tcp should further
our understanding of the molecular logic underlying ligand
recognition in this family of receptors.
The residues involved in ligand binding in Tar and Tsr (Fig.
1A and B) are located in the two helices (␣1 and ␣4) that
extend through the cytoplasmic membrane as the first and thesecond transmembrane regions (TM1 and TM2), respectively(28, 40). In Tar, three Arg residues (residues 64, 69, and 73)
within the helix ␣1 interact with the ␣- or ␤-carboxyl groups of
aspartate (40) (Fig. 1A and B). The corresponding Arg residues of Tsr are predicted to interact with the ␣-carboxyl or thehydroxyl group of serine (19, 22) (Fig. 1A and B). This triplet
is perfectly conserved in Tcp as residues 63, 68, and 72 (Fig.
1B), which are expected to interact with the carboxyl groups or

the hydroxyl group of citrate. Tyr-149 of Tar, which is locatednear the top of the helix ␣4 and interacts with the carboxylgroups of the ligand via water molecules (40), is also conservedin Tcp as Tyr-150. Because citrate has no amino group (Fig.
1C), it is reasonable that the Thr residue (154 in Tar and 156
in Tsr), which interacts with the amino group of aspartate orserine, is not conserved in Tcp (39). Since citrate has a polarhydroxyl group and three carboxyl groups (Fig. 1C), it is likelythat additional polar or positively charged residues in Tcp are
involved in the interaction with citrate.
As an initial attempt to understand the citrate-recognition
mechanism, we substituted Cys for seven polar or basic residues (Arg-63, Arg-68, Arg-72, Lys-75, Arg-78, Tyr-150, andLys-157). Plasmids encoding the resulting mutant proteins
were introduced into the E. coli strain HCB339 (⌬MCP) (37),which lacks all four chemoreceptors.
We first examined general receptor functions of the Cysreplaced Tcp proteins by swarming assay (Fig. 2). HCB339
cells expressing Tcp swarm in tryptone semisolid agar which
does not contain citrate (32). Although the actual stimulus towhich they respond is unknown, mutations in the C-terminal
methyltransferase-binding sequence of Tcp affect swarming
without impairing the citrate-sensing ability (32), demonstrating that this swarming requires the normal function of Tcp.
HCB339 cells expressing any mutant Tcp receptor swarmed as
fast as those expressing wild-type Tcp, suggesting that all of themutant receptors retain general receptor functions including
signaling and adaptation.
We then examined receptor capabilities of the Cys-replaced
Tcp proteins by temporal stimulation assay as described previously (31). Without chemotactic stimulation, cells expressing
wild-type or any mutant Tcp swam smoothly. When 15% glycerol was added, cells expressing each mutant Tcp showed tumbling responses similar to those of cells expressing wild-type
Tcp (data not shown), indicating that all of these proteins are
expressed and retain the ability to mediate repellent responses
to glycerol. Fig. 3A shows the citrate-sensing properties of themutant Tcp proteins. The R78C and K157C receptors conferred the same citrate-sensing ability as wild-type Tcp. The
R72C, K75C, or Y150C receptor mediated responses to citratethat were weaker than those mediated by wild-type Tar: 50 mM
citrate was required for the 50% smooth-swimming fraction ofcells expressing any of these mutant receptors, whereas a concentration of 1 mM is enough for that of cells expressing
wild-type Tcp. HCB339 cells expressing Tcp-R63C or Tcp-

* 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@nucc.cc.nagoya-u.ac.jp.
† Present address: Department of Clinical Pathology, Division of
Molecular Cytogenetics, International Medical Center of Japan, 1-21-1
Toyama, Shinjuku-ku, Tokyo 162-8655, Japan.
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NOTES

J. BACTERIOL.

FIG. 1. Ligand-binding sites of the bacterial chemoreceptors. (A) Schematic illustration of the ligand-binding residues of Tar and Tsr. The hydrogen bonding
pattern of the aspartate binding site of Tar was drawn according to Yeh et al. (40). It should be noted that the backbone carbonyl atoms of Gln-152, Phe-150, andTyr-149 are involved in hydrogen bonding. The serine-binding site of Tsr was deduced from the sequence similarity with Tar. Residues shown in ovals are from one
subunit and those in boxes are from the other. W, a bound water molecule. (B) Alignment of the amino acid sequences of the bacterial chemoreceptors of E. coli
(indicated with the subscript E) and Salmonella serovar Typhimurium (indicated with the subscript S). Bold letters indicate the residues involved in ligand recognitionin Tar and Tsr. Underlined residues of Tcp indicate those replaced by Cys in this study. Numbering of residues is for Tcp. (C) Structures of the attractants specific for
Tar, Tsr, or Tcp. The attractants are shown in their fully charged forms.

R68C showed no response to citrate up to a concentration of
50 mM. These results suggest that the residues Arg-63, Arg-68,
Arg-72, Lys-75, and Tyr-150 are important for sensing citrate.
We next examined the methylation patterns of the mutant
receptors by immunoblotting with anti-receptor serum (Fig.
3B) as described previously (32). Tcp is methylated at multiple

FIG. 2. Swarming abilities of HCB339 (⌬MCP) cells expressing wild-type(WT) and mutant Tcp receptors in tryptone semisolid agar which does notcontain citrate. Aliquots (1 ␮l each) of fresh overnight cultures were inoculated
onto tryptone semisolid agar (1% tryptone, 0.5% NaCl, 0.3% agar) supplemented with 25 ␮g of chloramphenicol per ml, and then the plate was incubatedat 30°C for 14 h.

residues in the cytoplasmic domain, and its methylation level
increases and decreases in response to citrate and glycerol,
respectively, to result in adaptation (39). Methylation and demethylation of a receptor can be detected as mobility shifts ofthe protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): the more the receptor is methylated, the faster it migrates in the gel (3, 4, 9, 10). Tcp-R63C
migrated a little faster than wild-type Tcp and the other mutant
Tcp receptors. However, the receptor does not seem to beproteolytic fragments but seems to be a full-length receptor: (i)Tcp-R63C expressed in HCB339 appeared as multiple bands
corresponding to differential levels of methylation (Fig. 3B),
whereas the same receptor expressed in a ⌬MCP strain lacking
the methyltransferase CheR and the methylesterase/deamidase CheB appeared as a single band with a mobility fasterthan that of wild-type Tcp (data not shown); (ii) Tcp-R63C was
detected with antiserum raised against the C-terminal 20-amino-acid sequence of Tar (data not shown), whereas the mutant
Tcp receptor lacking the C-terminal residue (Phe-547) was not(H. Okumura, M. Homma, and I. Kawagishi, unpublished results); and (iii) the corresponding mutant (R64C) Tar protein
also migrates faster than wild-type Tar (data not shown).
In the absence of citrate, all of the mutant proteins showed
methylation patterns similar to that of wild-type Tcp. All of themutant receptors were demethylated in response to the addition of the repellent glycerol. This result indicates that all of
the mutant receptors retain general signaling and adaptation
abilities. In the presence of citrate, the receptors which mediated attractant responses to citrate (Tcp-R78C and TcpK157C) showed elevated methylation levels. In contrast, citrate did not influence the methylation levels of Tcp-R63C and
Tcp-R68C, which failed to mediate responses to citrate andincreased only marginally those of Tcp-R72C, Tcp-K75C, andTcp-Y150C, which mediated weaker responses. These results

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1439

FIG. 3. Citrate-sensing abilities of the Cys-replaced Tcp receptors. (A) Temporal stimulation assay of cells expressing the Cys-replaced Tcp receptors.
HCB339 (⌬MCP) cells expressing wild-type or mutant Tcp as the sole chemoreceptor were treated with 15% glycerol and then stimulated with various concentrations of citrate. The fraction of smooth-swimming cells after 30 s wasmeasured at 25°C as described previously (31). Symbols: open circles, wild-type
Tcp; closed circles, Tcp-R63C; open triangles, Tcp-R68C; closed triangles, TcpR72C; open squares, Tcp-K75C; closed squares, Tcp-R78C; open diamonds,
Tcp-Y150C; closed diamonds, Tcp-K157C. (B) Immunoblotting analysis ofmethylation patterns of the Cys-replaced Tcp receptors. HCB339 (⌬MCP) cells
expressing wild-type or mutant Tcp were incubated with 15% glycerol (Glyc),
distilled water (None), 10 mM citrate (Cit-10), or 50 mM citrate (Cit-50) at 25°C
for 30 min. Samples were subjected to SDS-PAGE followed by immunoblottingwith anti-Tsr serum (18), which cross-reacts with Tcp, as described previously(32).

indicate that the R63C, R68C, R72C, K75C, and Y150C receptors are fully or partially defective in citrate-stimulated
methylation, corresponding well to the defects in behavioral
responses to citrate, and that the latter defects are not indirect
results from elevated levels of methylation, which would bias
the unstimulated behavior toward tumbling.
Two SH groups in close vicinity can form a disulfide bondunder moderately oxidizing conditions. Indeed, in some Cysreplaced mutants of Tar, the two subunits of the homodimerare cross-linked by a spontaneously formed disulfide bond(27). Thus, the defects in citrate sensing could be due to indirect effects of disulfide cross-linking. We therefore looked forintersubunit disulfide bonds in the Cys-replaced Tcp proteins.
After nonreducing SDS-PAGE followed by immunoblotting,bands with apparent molecular masses of about 60 kDa weredetected for all samples (Fig. 4A). In addition, with the R68C,
K75C, and R78C receptors, we found additional bands withapparent molecular masses of about 130 kDa (Fig. 4A). Thesebands disappeared by the treatment of the samples with 10%
2-mercaptoethanol prior to SDS-PAGE (Fig. 4A). Therefore,
we concluded that these bands represent disulfide-cross-linked
homodimers of the mutant Tcp proteins, suggesting that residues 68, 75, and 78 of one subunit of the Tcp homodimer are
located near the same residues of the partner subunit. This
result corresponds well to a configuration in Tar revealed by acomprehensive survey of disulfide cross-linking in Cys-scanned

mutant proteins (7): Ser-68 and Met-75 of the Tar homodimer
lie at the interface of helices ␣1 and ␣1⬘. In Tcp, the positional
equivalents are Asn-67 and Leu-74, respectively. If Tcp has asimilar helical structure, residues 68, 75, and 78 would belocated at positions adjacent to the interface, and residue 72
would be in the opposite faces. Moreover, in the case of TcpR78C, which had normal sensing abilities, both the crosslinked and the non-cross-linked species showed increases anddecreases in the methylation level in response to citrate andglycerol, respectively (Fig. 4B). Even in the case of R68C andK75C, which were defective in citrate sensing, the methylationlevels of the cross-linked dimers decreased in response to glycerol (Fig. 4B). This finding suggests that Tcp forms a functional
homodimer regardless of ligand occupancy states, as demonstrated for Tar (7, 8, 12, 24, 29), Tsr (23), and Trg (1, 16, 20).
For R68C, K75C, and R78C, the fractions of the crosslinked dimers were 0.2, 0.6, and 0.5, respectively. Thus, substantial amounts of uncross-linked homodimers are always
available, even for mutant receptors that undergo cross-linking. Moreover, the addition of dithiothreitol (up to 50 mM) did
not improve citrate responses of HCB339 cells expressing anyof these receptors (data not shown). Therefore, it is likely thatloss of the positively charged side chains of Arg-68 and Lys-75
itself impairs the ligand-binding affinity of Tcp for citrate.
Taken together, the data suggest that the residues Arg-63,

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J. BACTERIOL.

FIG. 4. Detection of disulfide-cross-linked dimers of the mutant Tcp proteins in nonreducing SDS-PAGE followed by immunoblotting. (A) Detection ofcross-linked dimers of Tcp. Ice-cold 5% trichloroacetic acid was added to HCB339 (⌬MCP) cells expressing wild-type (WT) or mutant Tcp proteins pretreated withor without 10 mM N-ethylmalemide (NEM). NEM was added to prevent disulfide formation during sample preparation. The NEM-pretreated or untreated samples
were collected by centrifugation and were dissolved in nonreducing SDS loading buffer supplemented with 10 mM NEM (lanes labeled with NEM) or 10%
2-mercaptoethanol (lanes labeled with 2ME), respectively. These samples were subjected to nonreducing SDS-PAGE followed by immunoblotting. (B) Methylation
patterns of cross-linked and uncross-linked Tcp proteins. HCB339 (⌬MCP) cells expressing wild-type or mutant Tcp were incubated with 15% glycerol (Glyc), distilledwater (None), 10 mM citrate (Cit-10), or 50 mM citrate (Cit-50) at 25°C for 30 min. After stimulation, the samples were treated with 10 mM NEM and 5%
trichloroacetic acid and were further treated as described above.

Arg-68, Arg-72, Lys-75, and Tyr-150 are involved in the recognition of citrate.
Tar has two rotationally symmetrical, antiparallel, nonoverlapping ligand-binding sites at the subunit interface (28, 40).
Arg-64 in one subunit of the Tar homodimer interacts with the
␣-carboxyl group of aspartate, and Tyr-149 in the same subunit
interacts with the ␣- and ␤-carboxyl groups via water molecules. Arg-69 and Arg-73 in the other subunit interact with the
␤-carboxyl group of aspartate. Presumably, some of the threecarboxyl groups or the hydroxyl group of citrate may interact
with Arg-68 and Arg-72 in one subunit of the Tcp homodimer
and with Arg-63 and Tyr-150 in the other subunit. Lys-75 may
also interact with some of the carboxyl groups and/or the hydroxyl group of citrate and may be one of the residues responsible for the ligand specificity, because this lysine is not conserved in Tar or Tsr. In contrast, the closely located basic
residue Arg-78 does not seem to be involved in ligand recognition. Another candidate for a determinant of ligand specificity is Lys-157. In Tar, the amino group of aspartate interacts
with Thr-154, which is not conserved in Tcp. Instead, Tcp has
the basic residue Lys-157 in this region. However, the substitution of Cys for Lys-157 did not affect citrate sensing at all.
Binding of aspartate to Tar does not cause a large rearrangement between TM1-␣1 and TM1⬘-␣1⬘ (5, 6, 7, 16, 20, 27) or
between TM1-␣1 and ␣4⬘-TM2⬘ (36) but triggers a slight axial
movement of ␣4-TM2 relative to TM1-␣1 and TM1⬘-␣1⬘ (6,16, 33). It is this movement that transmits information about
the extracellular binding event to the cytoplasmic signaling
domain. Based on the homology of Tcp with Tar, it is assumed
that residues 63, 68, 72, and 75 of Tcp are located at the apexof ␣1 and that residue 150 is near the apex of ␣4. Tcp would
seem to transduce signals via a similar process. However, ourresults also imply a possible difference between Tcp and Tar.
The critical movement of ␣4 in Tar presumably involves Thr154 (Thr-156 in Tsr), since it is a major contact with the ligandin ␣4. Lys-157 would play a similar role in Tcp, but unexpectedly the K157C mutant was normal for citrate taxis.
In this study, we targeted several residues in the putative

ligand-binding regions for Cys replacement. The mutants canbe further characterized by chemical modification as has beensuccessfully applied to Tar (14, 15) and Tsr (17, 18). Thesepolar and positively charged residues were chosen for mutagenesis, based on the homology of Tcp with the well-characterized chemoreceptors Tar and Tsr. Among the residues atwhich Cys substitutions disrupted citrate taxis, Arg-63, 68 and
72 are conserved in Tar and Tsr and Tyr-150 is conserved inTar. Lys-75 is the only residue unique to Tcp. It is likely thatsome other residues also interact with citrate. Further experiments, such as random mutagenesis, are needed to elucidate
the precise molecular mechanism underlying the recognition of
citrate, including discrimination between citrate and a metal
ion-citrate complex.
We thank Michael D. Manson of Texas A & M University forcritically reading the manuscript.
This work was supported in part by grants-in-aid for scientific research to I.K. from the Ministry of Education, Science, Sports andCulture of Japan and from the Takeda Science Foundation.
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