JOURNAL OF BACTERIOLOGY, Apr. 1995, p. 2218–2221
0021-9193/95/$04.0010
Copyright q 1995, American Society for Microbiology

Vol. 177, No. 8

In Vivo Sulfhydryl Modification of the Ligand-Binding Site ofTsr, the Escherichia coli Serine Chemoreceptor†
TOMONORI IWAMA,‡ IKURO KAWAGISHI,* SUMIKO GOMI,§ MICHIO HOMMA, AND YASUO IMAE
Department of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan
Received 6 September 1994/Accepted 28 January 1995

The Escherichia coli chemoreceptor Tsr mediates an attractant response to serine. We substituted Cys for
Thr-156, one of the residues involved in serine sensing. The mutant receptor Tsr-T156C retained serine- andrepellent-sensing abilities. However, it lost serine-sensing ability when it was treated in vivo with sulfhydrylmodifying reagents such as N-ethylmaleimide (NEM). Serine protected Tsr-T156C from these reagents. We
showed that [3H]NEM bound to Tsr-T156C and that binding decreased in the presence of serine. By pretreating cells with serine and cold NEM, Tsr-T156C was selectively labeled with radioactive NEM. These results are
consistent with the location of Thr-156 in the serine-binding site. Chemical modification of the Tsr ligandbinding site provides a basis for simple purification and should assist further in vivo and in vitro investigationsof this chemoreceptor protein.
binding can be specifically inactivated in vivo, Tsr function canbe analyzed in its native setting.
Effects of the Thr-156-to-Cys replacement on receptor functions. This site-directed replacement (Thr-1563Cys) was carried out as described previously (19) with the tsr-carrying plasmid pJFG5 (24). The mutant gene and its product are referredto hereafter as tsr-T156C and Tsr-T156C, respectively. The
mutant gene tsr-T156S was described previously (19). Plasmids
that carried tsr, tsr-T156S, and tsr-T156C were transferred into
strain HCB339 (40), which lacks all four receptors. Wild-type
Tsr, Tsr-T156S, and Tsr-T156C had comparable glycerol-sensing abilities (Fig. 1). The serine concentration required for
one-half of the maximal attractant response was 2 mM for cells
that expressed wild-type Tsr or Tsr-T156S and 40 mM for cells
that expressed Tsr-T156C (Fig. 1). These results indicate thatthe replacement of Thr-156 (or Ser-156) with Cys, i.e., thechange from OH to SH, does not drastically affect Tsr receptor
functions, including serine-sensing ability.
SH modification and serine protection of Cys-156. SH modification and chemotaxis assays were carried out as described

Escherichia coli has four closely related chemoreceptors (forreviews, see references 3, 10, 22, 25, 31, 35, and 36); eachconsists of an N-terminal periplasmic ligand recognition domain, a C-terminal cytoplasmic signaling domain, and two
transmembrane segments. Each one detects various chemical
attractants in a highly sensitive and specific manner (12). For
instance, mutants that lack the serine receptor (Tsr) but retain
the aspartate receptor (Tar) do not respond to serine.
The initial steps toward elucidating the mechanism that underlies this precise molecular recognition were to isolate andcharacterize mutant receptors which were defective in attractant detection but retained other sensing abilities, such as repellent- (14, 30, 34, 38, 39) and temperature-sensing (13, 23,29) abilities. In Tar, Arg-64, Arg-69, Thr-154, and several otherresidues have been implicated in aspartate sensing (7–9, 18, 19,
28, 41). The crystal structure of the ligand-binding domain of
Salmonella typhimurium Tar has been determined in the presence and absence of aspartate (26, 33, 42). In the cocrystal,
aspartate is intercalated between the two subunits of the Tar
homodimer, and only one of the two nonoverlapping aspartate-binding sites is filled.
In contrast, Tsr has not been extensively studied in terms ofligand binding since earlier characterizations of mutants (11,20). Recently, isothermal titration calorimetry (21) and computer simulation (15) have been used to analyze ligand binding
by Tsr. Site-directed sulfhydryl modification analyses should
provide valuable information complementary to these studies,as they were successfully used to investigate the aspartatebinding site in Tar (8, 9). Therefore, we substituted Cys for
Thr-156, which has been implicated in serine sensing. If ligand* Corresponding author. Mailing address: Department of Molecular
Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya
464-01, Japan. Phone: 81-52-789-2993. Fax: 81-52-789-3001. Electronic
mail address: g44416a@nucc.cc.nagoya-u.ac.jp.
† This article is dedicated to the memory of Yasuo Imae, our groupleader, who died unexpectedly of a cerebral hemorrhage on July 2,
1993, at the age of 53, with deep sorrow, respect, and gratitude.
‡ Present address: Faculty of General Education, Gifu University,
Yanagido, Gifu 501-11, Japan.
§ Present address: Department of Entomology, University of California, Davis, CA 55616.

FIG. 1. Serine-sensing abilities of mutant Tsr receptors. HCB339 cells that
expressed wild-type Tsr (open circles), Tsr-T156S (closed circles), or Tsr-T156C(open triangles) were pretreated with 1 M glycerol and then stimulated withvarious concentrations of serine. The changes in smoothly swimming cells after
30 s were measured. All manipulations were done at 258C.

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FIG. 3. Protective effect of serine on NEM modification of Tsr-T156C. At
the time indicated by the arrow, 40 mM NEM was added to HCB339 cells that
expressed Tsr-T156C in the absence (open circles) or presence of 1 mM serine(closed triangles) or aspartate (closed circles). After serine or aspartate waswashed out, serine responses were measured as described in the legend to Fig. 2.

FIG. 2. Inactivation by sulfhydryl-modifying reagents of the serine-sensing
ability of Tsr-T156C. Various concentrations of NEM (A) or DTNB (B) wereadded at the time indicated by the arrow in each panel to cells that expressed
Tsr-T156C (closed symbols). Cells that expressed Tsr-T156S (open circles) weretreated with 50 mM NEM (A) or 200 mM DTNB (B). At intervals, aliquots weretaken and serine responses were measured by adding 10 mM serine as describedin the legend to Fig. 1. (A) Closed circles, 30 mM; closed triangles, 40 mM; closed
squares, 50 mM. (B) Closed triangles, 100 mM; closed circles, 200 mM.

previously (8). In the presence of 50 mM N-ethylmaleimide(NEM), the serine-sensing ability of Tsr-T156C decreased
drastically, whereas that of Tsr-T156S decreased only slightly(Fig. 2A). Glycerol-sensing abilities were not affected by NEM.
5,59-Dithiobis-2-nitrobenzoic acid (DTNB) produced similareffects (Fig. 2B). The inhibitory effect of DTNB was reversedby the reductant dithiothreitol (1 mM; data not shown).
If Thr-156 is in the serine-binding site, excess serine should
protect Tsr-T156C from SH modification. NEM inactivation of
Tsr-T156C was delayed drastically by the prior addition of 1
mM serine (Fig. 3). Even after 60 min of incubation, more than
one-half of the cells showed serine responses. Such a protective
effect was not observed with 1 mM aspartate. Furthermore,
serine, but not aspartate, had a similar protective effect on
DTNB modification of Tsr-T156C (data not shown). Since Tsr
has no other Cys residue (4), these results suggest that Cys-156
is located in the serine-binding site.
More than 200 mM DTNB is required for complete inactivation within 30 min of the serine-sensing ability of Tsr-T156C,
whereas 5 mM DTNB is enough to inactivate the aspartatesensing ability of Tar-T154C (8). On the other hand, nearly
equal concentrations of NEM inhibit the attractant-sensing
abilities of these two receptors. The ligand-binding pocket of

Tar may be a little larger than that of Tsr, reflecting theirligand sizes. If so, DTNB, a bulkier reagent than NEM, might
have easier access to the Cys residue of Tar-T154C than to thatof Tsr-T156C.
The lower the concentration of NEM or DTNB, the longer
the duration before there is a detectable decrease in the serine
response (Fig. 2). Therefore, the cell’s full response to serine
may require many fewer Tsr molecules than those expressedfrom multicopy tsr genes. Whether this is the case in the wild
type, which contains about 2,000 molecules of Tsr per cell (35),
may be examined by radioactive labeling.
Specific binding of radioactive NEM to Tsr-T156C. We constructed plasmid pGAN1, which contains tsr-T156C under thecontrol of the tac promoter and the lacIq gene, by recombining
the tsr-T156C-carrying plasmid pJUN11 and the tsr-overproducing plasmid pCS20 (32). HCB339(pGAN1) and HCB339(pCS20) cells produced comparable sizes of swarm in tryptone
semisolid agar (data not shown). However, in the presence of
200 mM DTNB, HCB339(pGAN1) produced a smaller swarm
than did HCB339(pCS20) (data not shown), indicating that
Tsr-T156C was expressed from pGAN1.
We examined the receptor proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) uponisopropyl-b-D-thiogalactopyranoside (IPTG) induction. To
avoid receptor methylation, which would have caused multiple
receptor bands on the gel, plasmids were transferred into
strain CP553 (5), which lacks CheB and CheR, the chemoreceptor-modifying enzymes. Fresh overnight cultures of cells
that carried pCS20 or pGAN1 were inoculated (1:100 dilution)
into LB medium (17) supplemented with 200 mg of ampicillin
per ml and grown at 308C. After 3 h, 1 mM IPTG was added
to each culture and cells were further incubated for 4 h. When
whole-cell extracts of both CP553(pGAN1) and CP553(pCS20)
were analyzed, the intensity of the Tsr band increased drastically after 4 h of induction, while those of the other bands
remained at the same levels (Fig. 4A). We have concluded thatcells that carry pGAN1 overproduce Tsr-T156C in the presence of IPTG.
We then examined NEM binding to Tsr-T156C. After fullinduction, cells were suspended in motility medium (20) andincubated with 50 mM [3H]NEM (0.3 Ci/mmol; Dupont, NEN
Research Products) for 30 min at room temperature. The

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J. BACTERIOL.

FIG. 5. Selective labeling of Tsr-T156C with radioactive NEM. CP553 cells
that overproduced wild-type Tsr or Tsr-T156C were pretreated with 50 mM cold
NEM in the presence (1) or absence (2) of 10 mM serine at 258C for 30 min.
After serine and NEM were washed out, cells were incubated with 50 mM[3H]NEM at 258C for 30 min. Tsr, CP553(pCS20); Tsr-T156C, CP553(pGAN1).

FIG. 4. Binding of radioactive NEM to Tsr-T156C and protective effect ofserine. (A) Overproduction of Tsr-T156C. CP553 cells that carried pCS20 (Tsr)or pGAN1 (Tsr-T156C) were grown in LB medium at 308C for 3 h (up to early
exponential phase) and further incubated for 4 h in the presence (1) or absence(2) of 1 mM IPTG. Total cell extracts were analyzed by SDS-PAGE. Tsr protein
is indicated by an arrow in each panel. (B) Binding of radioactive NEM to
Tsr-T156C. CP553 cells that overproduced Tsr-T156C were suspended in motility medium supplemented with various concentrations of serine or aspartate andincubated with 50 mM [3H]NEM at 258C for 30 min. Samples were subjected to
SDS-PAGE and fluorography.

reaction was terminated with 5% trichloroacetic acid, and thenprecipitates were subjected to SDS-PAGE and fluorography(37). The band with an apparent molecular mass of about 60
kDa, i.e., Tsr-T156C, was labeled strongly in the samples ofcells that expressed Tsr-T156C (Fig. 4B), whereas no corresponding band was detected in the samples of cells that expressed Tsr (data not shown). The intensity of the NEMlabeled 60-kDa band (Tsr-T156C) decreased as the serine
concentration increased (Fig. 4B). In contrast, 100 mM aspartate and 1 M glycerol had no effect. The intensity of the 45-kDa
band, which was observed in all samples, was not affected by
the addition of serine. Thus, serine competes with NEM only
for Tsr-T156C. These results are consistent with the effect of
NEM on serine-sensing ability and therefore indicate that Cys156 (hence, Thr-156 in wild-type Tsr) is located in the serinebinding site.
Interestingly, DTNB did not inhibit cell growth and motility(8). However, disulfide bond formation in FlgI, the subunit
protein of the flagellar basal body P ring, is essential for flagellation (6). The periplasmic protein DsbA (2, 16) and thecytoplasmic membrane protein DsbB (1, 27) are responsible
for this process (6). Therefore, critical Cys residues in FlgI,
DsbA, and DsbB may not be readily exposed to DTNB attack.
On the other hand, NEM inhibits cell growth (8). We haveshown that NEM binds to a considerable number of proteins(Fig. 4B and 5). It would be interesting to compare NEMbinding proteins and DTNB-binding proteins.
Selective modification of Tsr-T156C with radioactive NEM.
Using the serine protection effect, we selectively labeled TsrT156C among all of the membrane proteins in intact cells.
Cells were incubated for 30 min with cold NEM in the presence of 10 mM serine. After being washed thoroughly, cells
were incubated with radioactive NEM. A protein with an apparent molecular mass of 60 kDa (Tsr-T156C) was labeledstrongly (Fig. 5). The only other labeled protein is one justbelow the 60-kDa band, which is probably a proteolytic product of Tsr-T156C, because it also was protected from NEM
modification by serine and its intensity increased upon prolonged incubation. Thus, Tsr-T156C was selectively labeled invivo with radioactive NEM.
This should provide the basis for a simple purification procedure for the chemoreceptor protein by SH-specific affinity
chromatography. The ligand-binding ability of the purified protein could be assayed by competition between the ligand andradioactive NEM, as is the case in vivo. Its further characterization would provide invaluable information about ligand
binding and other aspects of the information processing of thisreceptor.
We thank H. C. Berg, J. Gebert, D. R. Graham, and C. Park forgenerously providing us with bacterial strains and plasmids. We areespecially obliged to J. S. Parkinson of the University of Utah for
critically reading the manuscript.
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