School of Molecular Biosciences, Washington State University,
Pullman, Washington 99164-4660
We extended characterization of mutational substitutions in the
ligand-binding region of Trg, a low-abundance chemoreceptor of
Escherichia coli. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class
of substitutions made the receptor insensitive, reducing ligand-induced
signaling, and another mimicked ligand occupancy, inducing signaling in
the absence of ligand. We tested these deductions with in vitro assays
of kinase activation and found that insensitive receptors activated the
kinase as effectively as wild-type receptors and that induced-signaling
receptors exhibited the low level of kinase activation characteristic
of occupied receptors. Differential activation by the two mutant
classes was not dependent on high-abundance receptors. Cellular context
can affect the function of low-abundance receptors. Assays of
chemotactic response and adaptational modification in vivo showed that
increasing cellular dosage of mutant forms of Trg to a high-abundance
level did not significantly alter phenotypes, nor did the presence
of high-abundance receptors significantly correct phenotypic
defects of reduced-signaling receptors. In contrast, defects of
induced-signaling receptors were suppressed by the presence of
high-abundance receptors. Grafting the interaction site for the
adaptational-modification enzymes to the carboxyl terminus of
induced-signaling receptors resulted in a similar suppression of
phenotypic defects of induced-signaling receptors, implying that
high-abundance receptors could suppress defects in induced-signaling
receptors by providing their natural enzyme interaction sites in
trans in clusters of suppressing and suppressed receptors.
As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of
phenotypic defects in induced-signaling forms of Trg involved
assistance in adaptation, not signaling.
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INTRODUCTION |
Transmembrane signaling and
interreceptor interactions of the receptors that mediate chemotaxis in
Escherichia coli and Salmonella typhimurium are
being studied extensively. This family of closely related receptors is
an attractive subject for such investigations because there is
extensive functional and structural information about these proteins
(15). X-ray crystallographic studies of water-soluble
receptor fragments (7, 24, 32), in combination with
cysteine and disulfide scanning of intact receptors (3, 4, 10,
13, 25, 26, 38), have provided a detailed model of the
three-dimensional organization of a native receptor (Fig.
1). In this model, the chemoreceptor
homodimer is an extended helical bundle in which the ligand-binding
site is located near the membrane-distal end of the periplasmic domain,
and the histidine kinase CheA and coupling protein CheW are associated
in a noncovalent complex at the opposite end of the bundle, over 350 Å away, at the membrane-distal end of the cytoplasmic domain.
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FIG. 1.
Chemoreceptor structure and positions of mutational
substitutions in Trg. The cartoon on the left shows a chemoreceptor
dimer as an extended helical bundle. Ovals near the middle of the
cytoplasmic domain mark methyl-accepting sites. On the right is a model
of the Trg periplasmic domain based on the structure of the Tar
periplasmic domain (32) and an alignment of chemoreceptor
sequences (Megan Peach, unpublished results). Below the model is the
amino acid sequence of the relevant segment of Trg with the extent of
helix  -1 indicated. Positions of induced-signaling (green) and
reduced-signaling (red) substitutions used in this study are indicated
with colored Corey-Pauling-K representations of the native side chains
in the structural model and by color in the sequence.
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Chemoreceptors.
Chemoreceptors function by controlling kinase
activity. Interaction of an unoccupied receptor with kinase activates
the enzyme, but binding of chemoattractant to the receptor lowers the
activity of the interacting kinase, causing a reduction in the cellular content of the phosphorylated form of response regulator CheY, a
resulting shift in the pattern of flagellar rotation, and, ultimately, an effect on motility. Ligand binding also activates a feedback loop of
sensory adaptation in which methylation of specific adaptational glutamyl residues in the receptor's cytoplasmic domain causes compensatory changes that restore kinase activity to its null, receptor-activated state. Thus, signaling from a ligand-binding site
has two effects on the other side of the membrane, a transient effect
on kinase activity and a persistent effect on receptor methylation.
Signaling neither causes (33) nor requires (12, 16,
26) dimer dissociation but instead appears to be an allosteric change within a stable dimer that initiates at the ligand-binding site,
traverses the membrane, and affects both methyl-accepting sites and the kinase.
Mutational substitutions near ligand-binding sites.
Residues
in and near ligand-binding sites would be expected to be involved in
initiation of conformational signaling. Evidence for such involvement
was provided by mutagenic analysis of a 20-residue ligand interaction
region of chemoreceptor Trg of E. coli (43), a
receptor that mediates taxis toward the attractants galactose and
ribose via recognition of two respective sugar-occupied, periplasmic binding proteins. The analysis used in vivo patterns of
adaptational methylation to identify two signaling phenotypes: (i)
insensitive, characterized by little or no increase in adaptational
methylation in the presence of attractant, suggesting reduced
sensitivity to ligand and thus reduced signaling; and (ii) mimic ligand
occupancy, characterized by increased adaptational methylation in the
absence of attractant, suggesting that the mutational substitutions
themselves mimicked the effect of ligand occupancy to induce signaling
and that the sensory system responded as usual to persistent signaling by a compensatory increase in adaptational methylation. Alignment of
chemoreceptor sequences (22) allows placement of the sites of these mutational substitutions in Trg on the known structure of the
periplasmic domain of Tar (7, 32). Substitutions that confer the insensitive phenotype are near the membrane-distal end of
helix -1 and in the solvent-exposed loop that extends from -1,
reasonable locations for changes that perturb effective ligand binding
(Fig. 1). Substitutions that mimic ligand occupancy are in the adjacent
segment of -1, deeper in the domain and packed on surrounding
helices, in locations at which altered side chains could influence the
relative positioning of interacting helices and thus induce signaling.
Low- and high-abundance receptors.
Trg is a low-abundance
receptor in E. coli, present in a wild-type cell at ~10%
the content of the two high-abundance receptors, Tsr and Tar
(19). In the absence of high-abundance receptors, methylation of Trg, adaptation to Trg-linked attractants, and chemotactic responses to those compounds are all detectable but inefficient (1, 17, 18, 20). These functional defects are
correlated with a crucial difference between high- and low-abundance receptors, a conserved, carboxyl-terminal pentapeptide, present only in
the high-abundance class, that interacts with both enzymes of
adaptational modification, the methyltransferase (42) and the methylesterase-deamidase (2). Grafting a
carboxyl-terminal sequence that carries the pentapeptide enzyme
interaction site onto Trg creates a low-abundance receptor that is
close to fully functional in methylation, adaptation, and mediation of
the tactic response in the absence of high-abundance receptors
(1, 18). This implies that high-abundance receptors
improve Trg function by providing the enzyme interaction site in
trans, creating an increased local concentration of
modification enzymes in clusters of heterologous receptors
(31). This notion is supported by the observations that,
in the absence of high-abundance receptors, adaptational methylation
and tactic efficiency of Trg are improved by overproduction of the
methyltransferase (37) and that, in vitro, the inefficient
methylation of a high-abundance receptor deleted for the enzyme
interaction site is enhanced by the presence of the same kind of
receptor carrying the site (27, 30).
Extending characterization of substitutions in the ligand-binding
region of Trg.
Transmembrane signaling initiated at a
ligand-binding site affects both methyl-accepting sites and kinase
activity. The mutational substitutions in the ligand-binding region of
Trg had been tested for effects on methyl-accepting activity
(43) but not for effects on kinase activity. In addition,
the mimicked occupancy receptors mediated taxis in one cellular context
(low dosage in the presence of high-abundance receptors) but not in
another (high dosage in the absence of high-abundance receptors)
(43). The origin of this difference had not been pursued,
but as we began to understand how high-abundance receptors assist
functional activities of low-abundance receptor Trg, we became
interested in investigating whether high-abundance receptors could also
suppress mutational defects in Trg. Thus, we extended characterization
of substitutions in the ligand-binding region of Trg, determining the
ability of insensitive and mimic-occupancy receptors to activate the
histidine kinase in vitro and assessing the influence of cellular
dosage and of high-abundance receptors on the function and signaling of
mutant receptors.
(Portions of this work were performed by B.D.B. in partial fulfillment
of the requirements for a Ph.D. in genetics and cell biology at
Washington State University.)
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MATERIALS AND METHODS |
Strains and plasmids.
CP177 (39), CP362
(39), and CP553 (9) are strains of
E. coli K-12, derived from OW1, that contain,
respectively, deletions of the chromosomal copies of trg;
tar, tsr, tap, and trg; or
tar, tsr, tap, trg,
cheB, and cheR. Mutational changes in
trg originally described and characterized by Yaghmai and
Hazelbauer (43) were transferred, using a 1,155-bp
CpoI-Eco52I fragment, from pMG2-derived plasmids
to pGB1 (9), which contains the trg coding
sequence fused to a tac promoter,
lacIq, and bla. Transfer of mutations
into a gene coding for Trg fused to the final 19 residues of Tsr was
accomplished by moving the same fragment into pAL75 (18).
All plasmid constructs were verified by DNA sequencing and transformed
into appropriate host strains.
In vivo assays.
Assays were performed with CP362 (lacking
high-abundance receptors) or CP177 (containing high-abundance
receptors) harboring pGB1-derived plasmids. Quantitative immunoblotting
demonstrated that growth of such strains in minimal medium without an
inducer resulted in a cellular dosage of Trg slightly lower than that produced from chromosomal trg expressed from its natural
promoter (~50%) (X. Feng and G. L. Hazelbauer, unpublished results).
Induction by 20 µM isopropyl-
-D-thiogalactopyranoside
(IPTG) resulted in cells containing sevenfold more Trg than was
produced from chromosomal trg (17), making the
cellular amount of the usually low-abundance receptor Trg comparable to
that of a high-abundance receptor. These two levels of induction were
used for both in vivo assays. Assays of chemotactic-ring formation in
semisolid agar were performed essentially as described by Hazelbauer et
al. (22). Plates containing 50 µg of ampicillin/ml were
inoculated with 1.5 µl (~ 7.5 × 105) of
logarithmic-phase, motile cells growing in tryptone broth containing
100 µg of ampicillin/ml, incubated for 12 h at 35°C in a humid
environment, and photographed with an Alpha Innotech digital camera.
To assay transmembrane signaling in vivo, we used long-term exposure to
attractant in a growing culture rather than stimulation by a temporal
gradient in order to reduce the possibility that the patterns used to
deduce features of transmembrane signaling would be affected by
significantly different rates of adaptation in different cellular
backgrounds (20). Four-milliliter volumes of H1 minimal
salts medium (21) containing required amino acids at 1 mM,
50 µg of ampicillin/ml, IPTG as appropriate for induction to a
high-abundance dosage (see above), and 20 mM sodium succinate or 20 mM
ribose were inoculated to ~2.5 × 107 cells/ml with
overnight cultures in tryptone broth containing 100 µg of
ampicillin/ml and incubated with agitation at 35°C. At a cell density
of ~2.5 × 108/ml, samples were removed and placed
in 10% trichloroacetic acid. Material from 2.5 × 107
cells was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis under conditions known to provide maximal resolution of
the various methylated species of Trg (11% acrylamide, 0.073%
bisacrylamide; pH 8.2) (23) and analyzed by immunoblotting with anti-Trg serum under conditions in which the intensity of staining
of the bands was a linear function of the amount of protein. Since
signaling assays involve comparison of cells grown in different cultures, the amount of Trg, and thus the intensities of the stained bands under unstimulated (succinate-grown) or stimulated (ribose-grown) conditions, were not always the same. However, for all the comparisons shown in the figures in this study, adjustment of the amount of sample
loaded on the gel confirmed that the differences seen in the figures
(for which the same amount of cellular material was loaded for all
strains and conditions) were not a function of the amount of stained
Trg in a sample.
For cells lacking high-abundance receptors, our anti-Trg serum was
sufficiently specific that Trg was the only protein visible in the
immunoblots of whole-cell samples. However, the antiserum reacts with
high-abundance receptors (36), consistent with extensive conservation of sequence in the cytoplasmic domains (5),
and thus there was a problem in specific detection of Trg in cells containing high-abundance receptors, the multiple forms of which have
electrophoretic mobilities very similar to those for the various forms
of Trg. For cells in which Trg was induced to a level of expression
equivalent to that of the high-abundance receptors, the problem could
be overcome since the antiserum has an approximately 10-fold higher
sensitivity for Trg than for Tsr or Tar (36), and thus we
could adjust the ratio of sample to antiserum to display only Trg bands
on immunoblots. At the low-abundance level of Trg expression in the
presence of high-abundance receptors, such a differential display was
not possible, and thus the modification-based signaling assay could not
be performed under this particular set of conditions.
In vitro kinase assay.
Trg-containing membranes were
prepared from CP553 cells harboring an appropriate plasmid and stored
at 
70°C (1). Trg content (usually ~10% of the total
protein) was determined by quantitative immunoblotting with purified
Trg as a standard, using an AlphaImager 950 digital camera system
(Alpha Innotech Corporation) and ImageQuant software (version 4.2;
Molecular Dynamics, Sunnyvale, Calif.). As described by Barnakov et al.
(1), CheA was mixed with the accessory protein CheW, the
response regulator CheY, and isolated membrane containing either no
receptor, wild-type Trg, or a mutant receptor. After incubation at room
temperature for 1 h to allow formation of receptor-kinase
complexes, radiolabeled ATP was added, and the reaction was stopped
after 5 s. Relative levels of phospho-CheY were determined by
SDS-polyacrylamide gel electrophoresis and phosphorimaging. Under the
conditions used, production of phospho-CheY was a direct reflection of
CheA activity.
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RESULTS |
Kinase activation by mutant forms of Trg.
Yaghmai and
Hazelbauer (43) interpreted patterns of in vivo covalent
modification to indicate that one class of mutational substitutions in
the ligand interaction region of Trg induced transmembrane signaling
and another class reduced signaling. We tested this notion by examining
activation of the CheA kinase in vitro by a representative set of these
mutant receptors, four from the insensitive class and four from the
mimic-ligand-occupancy class (Fig. 2). A
substituted receptor characterized as insensitive because it exhibited
little or no change in adaptational methylation upon stimulation with
ligand might do so because its cytoplasmic domain was generally
disrupted and thus not available for effective adaptational
modification or because it was specifically and locally perturbed near
the ligand-binding site. Interaction of CheA and the coupling protein
CheW with wild-type Trg increases kinase activity approximately
100-fold over its low level in the absence of receptor
(1). All four insensitive receptors provided approximately the same substantial activation (Fig. 2), indicating that their cytoplasmic domains were indistinguishable from that of the wild type
in this function and supporting the notion that those mutant receptors
were perturbed only in the region of the ligand-binding site near the
sites of substitution. Unfortunately, we could not extend the
characterization to effects of ligand occupancy on kinase activity in
the in vitro system because the very weak affinity of Trg for its
binding protein ligands (dissociation constants, ~0.5 mM
[44]) made addition of saturating ligand technically infeasible.
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FIG. 2.
Activation of kinase by wild-type and mutant forms
of Trg. Membranes containing no chemoreceptors (none),
wild-type Trg (WT), or a mutant form of Trg (designated by the
wild-type amino acid, residue number, and replacing amino acid) were
assayed for activation of CheA by measuring levels of phospho-CheY in a
coupled system. Values were normalized to those for the wild-type
receptor in the same experiment and are averages of six determinations
(two trials each on three different membrane preparations) ± standard error. Labels at the bottom of the figure indicate
induced-signaling (INDUCED) or reduced-signaling (REDUCED) forms of
Trg.
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If mutational substitutions that increase in vivo receptor methylation
in the absence of ligand induce the same conformational signaling as
ligand binding, then receptors containing those substitutions should
exhibit in vitro the same reduced activation of kinase characteristic
of ligand-occupied receptors (6). Alternatively, the
substitutions might alter the receptor in a way that results in
increased methyl-accepting activity without reducing kinase activation,
as observed in some cases of cysteine-substituted and oxidatively
cross-linked forms of receptor (11). All four substitutions thought to mimic ligand occupancy reduced kinase activation by Trg (Fig. 2). Thus, those substitutions in the
periplasmic domain had a transmembrane effect on the activity of the
cytoplasmic domain. The effect was the same as if ligand were bound. It
seems unlikely that this effect is a nonspecific disruption of the
domain on the other side of the membrane since the same substitutions increase the in vivo methyl-accepting activity of the cytoplasmic domain. In vitro, the transmembrane mutational effect might be to
reduce activation without perturbing the complex with the kinase, the
common view of the way ligand occupancy affects the enzyme (15), or it might affect complex assembly-disassembly, a
recently noted effect of ligand occupancy (29). In either
case, the substitutions in the periplasmic domain have transmembrane
effects on the cytoplasmic domain that mimic those created by ligand
occupancy. Taken together, the in vitro and in vivo data prompt us to
refer to the mutant proteins as reduced-signaling and
induced-signaling receptors, respectively.
Signaling mutants in different cellular contexts.
The original
study of mutational substitutions in the ligand-binding region of Trg
indicated that the ability of mimicked-occupancy receptors to mediate
chemotaxis was a function of cellular context (43). Cells
containing chromosomal copies of the genes for the high-abundance
receptors and of trg coding for a mimicked-occupancy mutant
exhibited significant Trg-mediated chemotaxis, but cells containing a
higher dosage of the same mutant Trg, produced from multiple copies of
a plasmid-borne gene, and lacking high-abundance receptors exhibited no
tactic response. This difference could have reflected effects of Trg
dosage, high-abundance receptors, or both. Thus, we investigated the
influence of receptor dosage and of high-abundance receptors on in vivo
activities of Trg proteins containing representative substitutions in
the ligand interaction region, six previously characterized as inducing
signaling and six known to reduce signaling. We utilized plasmid-borne
copies of trg under the tight control of a modified
lac promoter-operator, appropriate levels of inducer, and
host cells containing or lacking high-abundance receptors to test
wild-type Trg and the 12 mutant receptors for their ability to mediate
chemotaxis in four different cellular contexts: (i) Trg at a
low-abundance dosage in cells lacking high-abundance receptors, (ii)
Trg at a high-abundance dosage in cells lacking high-abundance
receptors, (iii) Trg at a low-abundance dosage in cells containing
high-abundance receptors Tar and Tsr, and (iv) Trg at a high-abundance
dosage in cells containing the two high-abundance receptors. To make
direct comparisons of responses in the four different contexts, we
assayed taxis by monitoring formation of chemotactic rings in semisolid
agar plates containing an attractant sugar as the sole source of carbon and energy. Of the available assays, only that for ring formation is
sufficiently sensitive to detect the weak Trg-mediated responses of
cells lacking high-abundance receptors (17). Experience
with many trg mutants has shown us that this assay is a
sensitive and reliable means of characterizing functional activities
and mutational defects in Trg (see, for instance reference
17). Cells performing Trg-mediated taxis form distinct
rings. The better the taxis, the faster the ring moves. Cells incapable
of Trg-mediated taxis form fuzzy disks lacking a distinct boundary. The
rate of movement of that disk is unrelated to the Trg-mediated
response. The mutant and wild-type proteins were assayed for
transmembrane signaling in vivo in all contexts except condition iii,
in which the low ratio of Trg to heterologous but structurally related
chemoreceptors prohibited specific detection of patterns of
adaptational modification of Trg by immunoblotting (see Material and Methods).
Mediation of taxis.
Altering the cellular content of the
wild-type (17) or mutant forms of Trg from their
characteristic low abundance level to a high abundance level had little
effect on their ability to mediate taxis toward galactose (Fig.
3A) or ribose (Fig. 3B). In Fig. 3, the
upper two rows and lower two rows of the panels each represent paired
sets of responses mediated by the wild type and each of the mutant
forms of Trg in cellular contexts that differed only in the dosage of
that receptor. In different cellular contexts and for different mutant
receptors, responses ranged from essentially wild type to not
detectable, but for any particular receptor a change in dosage from low
abundance (upper member of the pair) to high abundance (lower member)
had little or no effect on ring sharpness or rate of movement. In
contrast, the presence of high-abundance receptors had a profound
effect on the functional ability of induced-signaling receptors. In
cells lacking high-abundance receptors, all induced-signaling receptors
were incapable of mediating formation of chemotactic rings in response
to galactose (Fig. 3A) or ribose (Fig. 3B). However, those same
receptors mediated formation of distinct rings in the presence of
high-abundance receptors (Fig. 3, lower two rows of each panel), the
only exception being response to galactose mediated by Trg-Q79L, a
receptor previously shown to exhibit a differential defect in responses
to the two Trg-mediated attractants (44). Substitutions
that reduce signaling can have equivalent and drastic effects on the
responses to both Trg-linked attractants, as seen for Q79P and A87P, or
can affect the two responses differentially (44), as
observed for G81W (which has a greater effect on the response to
galactose) as well as for A82P, R85L, and R85S (which have a greater
effect on the response to ribose). The former situation would occur if
the altered side chain participated equally in productive interaction
with both sugar-binding proteins recognized by Trg, and the latter would occur if the side chain were more important for one of the interactions. Responses mediated by reduced-signaling receptors that
were undetectable in the absence of high-abundance receptors remained
undetectable in their presence, a pattern quite different from
the major improvement in response observed for the induced-signaling receptors. Detectable responses mediated by reduced-signaling receptors in the absence of high-abundance receptors were
quantitatively improved by the presence of the high-abundance
receptors, paralleling the effect observed for wild-type Trg
(17).
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FIG. 3.
Chemotaxis mediated by wild-type and mutant forms of
Trg. Shown are representative responses mediated by Trg in its
wild-type (WT) or signaling-mutant forms (designated as in the legend
to Fig. 2) expressed from plasmid-borne genes. Cells were assayed for
the ability to form chemotactic rings on semisolid agar plates
containing galactose (A) or ribose (B). Assays were performed on cells
lacking or containing the high-abundance receptors (designated in the
first column as "no" or "yes," respectively) and containing
each respective form of Trg expressed to a level characteristic of a
low- or high-abundance receptor (designated in the second column as
"low" or "high," respectively). Images were recorded 12 h
after inoculation and incubation at 35°C. Labels at the bottom of the
figure indicate wild-type (WT), induced-signaling (INDUCED), or
reduced-signaling (REDUCED) forms of Trg.
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Signaling phenotypes.
We assessed effects of cellular context
on the signaling phenotype by using SDS-polyacrylamide gel
electrophoresis and immunoblotting to determine in vivo patterns of
adaptational modification (43). In immunoblots, Trg
appears as an array of bands corresponding to different numbers of
covalent modifications per polypeptide chain (34, 35).
Receptors with glutamates at all methyl-accepting sites migrate most
slowly, and, with the exception of one methyl-accepting position among
the five in Trg, each added methyl group results in a characteristic
increase in electrophoretic mobility. As in other chemoreceptors, two
methyl-accepting glutamates in Trg are created by deamidation of
gene-encoded glutamines (35). Deamidation is catalyzed by
the methylesterase and controlled by the same factors as demethylation.
An amide group has the same effect on electrophoretic migration as a
methyl ester, resulting in a more rapidly migrating species
(35). Figure 4 shows
representative examples of signaling assays, comparing unstimulated and
ribose-stimulated cells, performed under the conditions used by Yaghmai
and Hazelbauer (43). For wild-type Trg (leftmost pair of
lanes), the pattern for unstimulated cells has four bands, designated
bands 1 (highest) through 4 (lowest), corresponding to a distribution
of, on average, ~1.5 methylated or amidated sites per receptor
(34, 35). In cells that have adapted to persistent
stimulation, methylation has increased and the pattern is dominated by
the fastest-migrating, most modified band (band 4) of the original set.
This shift indicates signaling from the periplasmic to the cytoplasmic
domain. Trg containing a substitution that induced signaling (Fig. 4,
middle pair) exhibits this shift even in the absence of ligand. In
contrast, the pattern of a reduced-signaling receptor (Fig. 4,
rightmost pair) is indistinguishable from that of the wild type in the
absence of ligand and is essentially unchanged in its presence.
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FIG. 4.
Signaling in vivo assessed in an immunoblot assay.
Modification state was used to assay transmembrane signaling in vivo by
a wild-type (WT), induced-signaling (INDUCED), or reduced-signaling
(REDUCED) form of Trg. Cells lacking chromosomal copies of
tsr, tar, tap, and trg but
harboring a plasmid coding for one of the forms of Trg were grown to
mid-log phase in minimal medium in the absence () or presence (+) of
excess ribose. Samples from each actively growing culture were analyzed
by SDS-polyacrylamide gel electrophoresis and immunoblotting with
anti-Trg serum. The forms of Trg were expressed at levels significantly
higher than the level characteristic of a high-abundance receptor
approximating the cellular content used in reference 43. The figure
shows the region of the immunoblots containing chemoreceptor bands; the
lowest band in the leftmost lane has an apparent
Mr of ~60,000.
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Cellular context had little influence on signaling assay patterns for
reduced-signaling receptors but had discernable effects on
induced-signaling receptors (Fig. 5). For
reduced-signaling receptors, the patterns observed in the absence of
stimulation were very similar to the pattern for unstimulated,
wild-type Trg and were shifted little, if at all, upon stimulation. The
greatest effect was for ribose stimulation of Trg-G81W, consistent with the ability of this mutant receptor to mediate a tactic response to
ribose. Neither changing the dosage of reduced-signaling receptors nor
introducing high-abundance receptors significantly altered either the
patterns obtained in the absence of stimulation or the lack of a
significant shift in the presence of ligand. For induced-signaling
receptors, signaling assay patterns in the absence of stimulation all
exhibited a characteristic shift toward more rapidly migrating, more
adaptationally modified bands (Fig. 5). The degree of shift, and
presumably the degree of signaling, correlated with the
particular mutational substitution. In all cellular contexts, stimulation of induced-signaling receptors by ligand resulted in an
additional shift to faster-migrating, more highly modified forms from
the already shifted pattern of the unstimulated receptor, indicating that induced-signaling receptors were capable of
transmembrane signaling in response to occupancy by their natural
ligands. Evidence for such signaling was not clear in the signaling
assays performed in the original study of these mutant receptors (see
reference 43 and the example in Fig. 4), probably because
those assays were done with cells containing receptor dosages at least
25-fold above a normal high-abundance dosage and thus had an abnormal receptor-modification enzyme stoichiometry. Increasing the dosage of
induced-signaling receptors from a low abundance to a high abundance
level resulted in only a modest shift to more-rapidly migrating
electrophoretic forms, consistent with the lack of a discernible effect
of dosage on the tactic response. In cells containing
high-abundance receptors, the electrophoretic patterns of
induced-signaling receptors all exhibited an increased number of
electrophoretic forms (Fig. 5, compare rows 2 and 4), indicating that
adaptational covalent modification was more active in the cells in
which the mutant receptors were able to mediate taxis. This correlation
is likely to reflect the crucial contribution of high-abundance
receptors in functionally assisting induced-signaling forms of Trg (see
Discussion).
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FIG. 5.
Signaling by wild-type and mutant forms of Trg. The
forms of Trg analyzed in the study shown in Fig. 3 were tested for
signaling in vivo, as described in the legend to Fig. 4, by
immunoblotting of cells grown in minimal medium in the absence () or
presence (+) of excess ribose. Assays were performed on cells lacking
or containing the high-abundance receptors (designated in the first
column as "no" or "yes," respectively) and containing each
respective form of Trg expressed to a level characteristic of a low- or
high-abundance receptor (designated in the second column as "low"
or "high," respectively). Recognition of conserved regions of the
high-abundance receptors by anti-Trg serum prohibited specific staining
of Trg expressed at a low-abundance level in cells containing the
high-abundance receptors. Labels are as for Fig. 3.
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Effects of the enzyme interaction site.
The inefficient taxis
and adaptational modification mediated by the wild-type
form of the low-abundance receptor Trg in cells lacking
high-abundance receptors are significantly improved by addition to Trg
of the 19-residue, carboxyl-terminal segment (from a high-abundance
receptor) that contains the pentapeptide site for interaction with the
enzymes of adaptational modification (1, 18). Since the
presence of high-abundance receptors greatly improved the function of
induced-signaling forms of Trg, we examined the effects of introducing
the interaction site at the carboxyl terminus of induced-signaling
receptors. Four such constructs were created and assayed for their
ability to mediate taxis and for patterns of covalent modification in
the signaling assay (Fig. 6). Grafting
the enzyme interaction site onto induced-signaling forms of Trg
conferred the ability to mediate taxis in the absence of high-abundance
receptors and resulted in an increase in the number of electrophoretic
forms seen in the immunoblot assay. Thus, the presence of the enzyme
interaction site exerted a corrective influence on induced-signaling
forms of Trg whether present in trans (on high-abundance
receptors) or in cis (grafted to the carboxyl terminus).
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|
FIG. 6.
Effects on induced-signaling forms of Trg of the
interaction site for the enzymes of adaptational modification provided
in trans or in cis. Abilities to mediate taxis
and signaling phenotypes of wild-type (WT) and four induced-signaling
forms of Trg are shown in cells lacking high-abundance receptors and,
thus, lacking the carboxyl-terminal sequence that interacts with the
enzymes of adaptational modification (none), in cells containing
high-abundance receptors and thus providing the interaction sequence in
trans relative to Trg (trans), and in cells
containing Trg proteins with carboxyl-terminal extensions comprising
the final 19 residues of the high-abundance receptor Tsr
(18) and thus having the interactions sequence in
cis relative to Trg (cis). Assays were performed
as detailed in the legends to Fig. 3 and 5 and were done on cells with
Trg expressed at a high-abundance dosage. Signaling assays were
performed with (+) or without () ribose.
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DISCUSSION |
In this study, we extended our investigation of mutational
substitutions in the ligand interaction region of chemoreceptor Trg to
in vitro assays of kinase activation and to characterization of the
effects of cellular context on mutational phenotype. Results of the
kinase assays supported the notion that the two classes of
substitutions induced or reduced signaling. The effects of cellular
context revealed that the phenotypic effects of one class of
substitutions were suppressed by heterologous, high-abundance receptors
that acted to enhance adaptational modification of the mutant,
low-abundance receptors, probably as the result of physical proximity
and receptor clustering
Signaling mutants.
The notion that substitutions in the ligand
interaction region of Trg induced or reduced signaling was based
exclusively on patterns of receptor modification in vivo
(43). An important test was to determine the effects of
the mutations on kinase activation, a determination that required an in
vitro assay. The results (Fig. 2) confirmed and strengthened the
definition of the two phenotypic classes, one that reduced signaling
and the other that induced signaling, and indicated that differential
activation of kinase by the two mutant classes was not dependent on
high-abundance receptors. The effects of cellular context were also
consistent with the two postulated classes of signaling mutants.
Reduction of signaling by disruption of effective ligand binding should not have been corrected by the presence of high-abundance receptors, and it was not (Fig. 3 and 5). In contrast, persistent signaling generated by a mutational substitution could disrupt the sensory system
if methylation were not sufficiently efficient to balance that
signaling. In this light, the inability of induced-signaling receptors
to mediate taxis in the absence of high-abundance receptors and the
much-improved function of the mutant receptors in their presence can be
readily understood as a reflection of the inefficiency of adaptational
modification in the former context and of its efficiency in the latter.
Since Trg is a low-abundance receptor, it was important to determine
whether the phenotypes of signaling mutants would be affected by
receptor dosage. We found that changing the cellular content of the
mutant forms of Trg from one approximating the natural dosage of a
low-abundance receptor to one approximating the dosage of a
high-abundance receptor had only modest effects for a few mutants. This
lack of a substantial effect of cellular dosage parallels observations
that the efficiency of wild-type Trg in mediating taxis is minimally
affected over a wide range of dosages (17). The mutant
receptors for which we observed modest improvement in function at a
higher cellular dosage were those with differential defects in
responses to galactose and ribose. These substitutions are likely to
reduce, but not eliminate, effective binding to the affected ligand
(44), so it is plausible that an ~10-fold increase in
receptor dosage could increase that interaction and thus improve taxis.
Covalent modification, signaling, and taxis.
The patterns
exhibited by reduced-signaling receptors in the signaling assay
correlated directly with the ability of these receptors to mediate
taxis. Five reduced-signaling receptors exhibited little, if any, shift
to more highly methylated forms in the presence of stimulating ribose,
and those same five were unable to mediate taxis toward that sugar. One
reduced-signaling receptor (Trg-G81W) exhibited detectable, although
reduced, signaling in response to ribose, and it mediated detectable
taxis. For the induced-signaling receptors, the situation was more
complicated. Signaling, as detected by a significant shift to
faster-migrating electrophoretic forms in the persistent presence of
attractant, was observed for all six induced-signaling receptors in all
cellular contexts that could be examined. However, these receptors
mediated taxis only in the presence of high-abundance receptors (Fig.
3) or when provided with a carboxyl-terminal tail carrying the
interaction site for the enzymes of adaptational covalent modification
(Fig. 6). For each induced-signaling receptor, effective mediation of
taxis was correlated with an increased number of different
electrophoretic forms in the signaling assay (Fig. 5 and 6). This can
be explained by a greater efficiency of covalent modification of
induced-signaling receptors in cells containing the enzyme interaction
site on heterologous receptors in a cluster or on the mutant receptor
itself. The reduced number of electrophoretic forms in the absence of
the enzyme interaction site is reminiscent of Trg with alanines at the
positions of glutamines usually deamidated by the
methylesterase-deamidase to create methyl-accepting glutamates
(34). The similarity suggests that the reduced number of
electrophoretic forms of induced-signaling receptors in the absence of
enzyme interaction sites reflects incomplete deamidation, resulting in
fewer methyl-accepting sites. It is likely that deamidation of Trg
would be incomplete because the reaction is inefficient in the absence
of the enzyme interaction site (2), and persistent inhibition of the kinase by mutational signaling would result in low
cellular levels of phosphorylated and, therefore, activated deamidase.
Slow and probably incomplete adaptational modification in the absence
of the enzyme interaction site is likely to be the origin of the
inability of induced-signaling receptors alone to mediate taxis. In the
absence of high-abundance receptors, wild-type Trg is capable of
mediating detectable taxis (17, 22) and of adapting to low
levels of receptor occupancy, although at a reduced rate (20). At higher levels of occupancy, adaptation times are
greatly extended and it appears that the native receptor, lacking an
enzyme interaction site, is not capable of becoming sufficiently
methylated to balance the signal generated by ligand binding
(20). This is likely the situation for the
induced-signaling receptors, in which the mutational substitutions
generate a signal too strong to be balanced by the relatively
inefficient methylation of a receptor lacking the enzyme interaction
site. By this reasoning, it is easy to understand the correlation
between the larger array of electrophoretic forms, an indication of
more efficient adaptational modification, with the ability of
induced-signaling receptors to mediate chemotaxis.
Functional interactions among heterologous receptors.
Recently
there has been much interest in chemoreceptor clustering
(31) and the possibility that clustering and physical interaction between receptors is crucial for the functions of receptor sensitivity, signaling, and signal amplification (8, 14,
24, 28). There are only a few documented examples of functional
interactions between receptors in vivo, and all concern the dependence
of low-abundance receptors on high-abundance receptors for effective
function (17, 18, 20, 40, 41). The present study extends
this set of data by providing examples in which high-abundance
receptors suppress, via adaptational improvement, mutational defects in
a specific class of mutant forms of the low-abundance receptor Trg.
This mutational suppression provided independent evidence for
adaptation-related, functional interaction between heterologous chemoreceptors.
In the absence of high-abundance receptors, the induced-signaling forms
of Trg were unable to mediate chemotaxis, but the presence of
high-abundance receptors made the mutant receptors functional (Fig. 3).
This was not a general effect on all types of Trg signaling mutants,
since the phenotype of reduced-signaling mutants was unaltered by the
presence of high-abundance receptors. The inability of
induced-signaling forms of Trg to mediate taxis was also corrected by
introducing the interaction site for the enzymes of adaptational
modification at the carboxyl terminus of the mutant receptors (Fig. 6).
This implies that suppression of the mutant phenotype of
induced-signaling receptors by high-abundance receptors was mediated by
provision of the enzyme interaction site naturally present at the
carboxyl terminus of high-abundance receptors. Enhanced
adaptational modification of mutant, low-abundance receptors by
the presence of high-abundance receptors carrying interaction sites for
the modification enzymes indicates that the heterologous receptors must
be in physical proximity. As for functional interactions in vivo
between wild-type forms of low-abundance and high-abundance receptors
(17, 18, 41), suppression of mutational defects in Trg by
high-abundance receptors implicates receptor interaction and clustering
(31) in adaptation rather than signaling.
In contrast, although signaling from a low-abundance receptor like Trg
must involve significant amplification to have an effect on cellular
behavior in the presence of an excess of high-abundance receptors,
there is as yet no evidence that signaling from the low-abundance
receptor Trg is dependent on other receptors. In cells lacking
high-abundance receptors, binding of ligand to Trg generates signaling
that effectively alters cellular behavior even though adaptation is
drastically inefficient (20). In vitro, Trg activates the
kinase in the absence of any heterologous receptor (1),
and the present study indicates that mutationally induced signaling
from the periplasmic domain of Trg reduces kinase activity without
assistance from high-abundance receptors. In summary, investigations of
both mutant and wild-type forms of low-abundance receptors emphasize
the importance of heterologous, high-abundance receptors for effective
adaptation, not for excitatory signaling. Receptor proximity and,
presumably, clustering have an important role in effective sensory
adaptation of low-abundance receptors. A role in signaling has yet to
be documented.
We thank Alexander Barnakov and Ludmilla Barnakova for purified
CheA, CheW, CheY, and Trg; Xiuhong Feng for guidance on the kinase
assay; Megan Peach for her model of the Trg periplasmic domain; and
Douglas Banks and Angela Lilly for introduction of signaling mutations
into the hybrid gene coding for Trgt and for initial characterization
of those constructs.
This work was supported in part by research grant GM29963 and
Biotechnology Training Grant T32 GM08336 from the National Institutes of General Medical Science.
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Abstract
Introduction
Columbia,
Columbia, MO 65211. Phone: (573) 882-4845. Fax: (573) 882-5635. E-mail: hazelbauerg{at}missouri.edu.
Present address: Department of Biology, California Institute of
Technology, Pasadena, CA 91125.
