From the Department of Biochemistry, Colleges of § Medicine and Liberal Arts and Sciences, University of Illinois, Urbana, Illinois 61801
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ABSTRACT |
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For the Gram-positive organism Bacillus
subtilis, chemotaxis to the attractant asparagine is mediated by
the chemoreceptor McpB. In this study, we show that rapid net
demethylation of B. subtilis McpB results in the immediate
production of methanol, presumably due to the action of CheB. We also
show that net demethylation of McpB occurs upon both addition
and removal of asparagine. After each demethylation event,
McpB is remethylated to nearly prestimulus levels. Both remethylation
events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl
transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested.
Furthermore, we show that the remethylation of asparagine-bound McpB
requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is
supported by two observations: a cheRBCD mutant is
capable of transient excitation and subsequent oscillations that bring
the flagellar rotational bias below the prestimulus value in the
tethered cell assay, and the cheRBCD mutant is capable of
swarming in a Tryptone swarm plate.
Chemotaxis is the process by which bacteria sense their chemical
environment and migrate toward more favorable conditions. In
Bacillus subtilis, chemotaxis toward the attractant
asparagine has been shown to be mediated by the methyl-accepting
chemotaxis protein McpB (1). When asparagine is added to membranes
containing McpB in vitro, the rate of autophosphorylation of
the CheA autokinase increases (2). The phosphorylated form of CheA
transfers a phosphoryl group to CheY to produce CheY-P (2, 3), which then interacts with switch proteins to cause
CCW1 rotation of
the flagella, resulting in smooth swimming behavior (3). CheA-P also
donates phosphoryl groups to
CheB,2 which
thereby becomes activated to demethylate the MCPs and produce methanol
(4, 5). Methylation of the MCPs is known to occur on glutamate side
chains (6) through the action of CheR, the chemotactic
methyltransferase, which utilizes AdoMet as a substrate (7).
The B. subtilis chemotactic machinery also includes CheW,
CheC, CheD, and CheV. CheW and CheV are thought to couple CheA activity to the MCPs (8-11). CheC inhibits methylation of the MCPs by an unknown mechanism but does not interfere with the methylesterase, CheB
(12, 13). CheD is required to produce a normal prestimulus bias, normal
methylation, and azetidine-2-COOH-induced activation of CheA in
vivo (12). How these proteins interact to regulate the chemotactic
response in B. subtilis remains unknown.
The chemotaxis system in Escherichia coli has been well
characterized and has served as a paradigm for our studies (for
reviews, see Refs. 14-17). The E. coli system includes
homologs of the MCPs, CheA, CheB, CheR, CheW, and CheY. The E. coli system also includes CheZ, which facilitates
dephosphorylation of CheY-P (18-21), but does not include homologs to
CheC, CheD, or CheV. Thus, the E. coli and B. subtilis chemotactic mechanisms must differ. Indeed, in E. coli repellent stimulation is thought to increase CheA activity (18, 22-24), and CheY-P is thought to interact with the flagellar switch to cause tumbling (25-27).
Regulation of methylation during chemotaxis has been shown to be more
complex in B. subtilis than in E. coli. Both
addition and removal of all amino acid attractants result in methanol
production (28), as was previously shown to be the case for aspartate
(29) and alanine (30). Methanol formation during chemotaxis in E. coli, however, increases in response to repellent stimuli and decreases in response to attractant stimuli (31, 32). Second, in
B. subtilis it has been hypothesized that an acceptor may
receive methyl groups from the MCPs during a period of increased
turnover due to attractant addition (29, 33-37). Finally, CheC and
CheD affect receptor methylation and behavior in B. subtilis
(12, 13), whereas no homologs to these proteins exist in E. coli.
In this study, we have tested the methyl transfer hypothesis in
B. subtilis by examining asparagine-induced methylation
changes on McpB. Our results demonstrate that no methyl group transfer need be postulated to occur during chemotaxis to asparagine in B. subtilis. The observed methanol production in response to addition and removal of asparagine (28) is due to two independent demethylation events on McpB catalyzed by CheB-P. After each demethylation event, McpB is remethylated by CheR using AdoMet as a substrate. We also show
that remethylation of asparagine-bound McpB requires the response
regulator CheY-P, suggesting that a feedback mechanism may exist to
bring about adaptation to positive stimuli. This hypothesis is
supported by behavioral data showing transient excitation and
subsequent oscillation in a cheRBCD mutant, which lacks all proteins previously thought to be involved in adaptation during chemotaxis in B. subtilis.
Bacterial Strains and Plasmids--
All bacterial strains and
plasmids used in this study are listed in Tables
I and II,
respectively. All plasmids were propagated in E. coli strain
TG-1 (Amersham Pharmacia Biotech).
Chemicals--
L-[methyl-3H]Methionine
(80-85 Ci/mmol) was obtained from Amersham Pharmacia Biotech. All
other chemicals were of reagent grade.
Solutions and Growth Media--
Luria-Bertani (LB) medium is 1%
Tryptone, 0.5% yeast extract, and 1% NaCl. Tryptone broth is 1%
Tryptone and 0.5% NaCl. Chemotaxis buffer (CB) is 0.1 mM
EDTA, 50 µM CaCl2, 0.05% glycerol, 5 mM sodium lactate, 0.3 mM ammonium sulfate, and
20 mM potassium phosphate, pH 7.0 (38). Minimal medium is
50 mM potassium phosphate, 1 mM
(NH4)2SO4, 1.2 mM
MgCl2, 140 µM CaCl2, 10 µM MnCl2, 50 µg/ml required amino acids,
and 20 mM sorbitol, pH 7.0 (38). Protoplast buffer is 20%
sucrose, 25 mM potassium phosphate, 10 mM
MgCl2, 30 mM sodium lactate, and 1.0 mM EDTA, pH 7.0 (39).
Construction of an amyE Integration Plasmid Carrying mcpB--
A
2.8-kb BglII-XbaI fragment containing
mcpB was subcloned from pDW12 (1) into pBluescript
(SK Construction of McpB Expressing Strains--
Four mcp
homologs (mcpB, tlpA, mcpA, and
tlpB) identified by Hanlon and Ordal (1) are located at
279° on the B. subtilis chromosome. A plasmid was
constructed such that the 5'-end of mcpB (about 300 bp) and
3'-end of tlpB (about 100 bp) were separated by a
cat cassette to create pMR134. Plasmid pMR134 was linearized and crossed onto the chromosome of the B. subtilis wild-type
strain (OI1085). CmR transformants were selected, thereby
creating strain OI3180, which has the four mcp homologs
deleted.4
Integration of mcpB into the amyE locus of OI3180
was achieved using linearized pUNK200 to create strain OI3184.
KanR transformants were selected, and potential
amyE integrants were screened by the starch hydrolysis test.
Colonies grown on tryptose blood agar base with 0.2% starch were
flooded with Mordant's solution: 0.33% I2, 0.67% KI in
H2O. Integrants, negative for starch hydrolysis, do not
form clear zones around the colonies (41). Chromosomal DNA from OI3280
(mcpC4::erm; 42) was then used to transform OI3184 cells, and erythromycin-resistant transformants were selected to
generate strain OI3290. OI3290 cells
(CmRKanREmRAmy
Strain OI3294 was constructed in a manner similar to strain OI3290. A
strain deleted for cheY (OI2057; 43) was transformed with
chromosomal DNA from strain OI3180 to generate strain OI3289 (CmR). This strain was then transformed with linearized
pUNK200, selected for KanR, and screened for integration at
the amyE locus by the starch hydrolysis assay to create
strain OI3292. OI3292 cells
(CmRKanRAmy Construction of the cheRBCD Mutant--
A plasmid was designed
to delete the adjacent B. subtilis genes, cheC
and cheD. A partially digested 1.8-kb
PstI-EcoRI fragment containing cheD
and the 5'-end of sigD (pMR116)4 was
cloned into the PstI-EcoRI sites of pUC18,
thereby creating plasmid pAIN500. The 1.8-kb fragment carried an
EcoRI site, introduced by site-directed mutagenesis, that is
internal to cheD and approximately 8 codons from the normal
stop codon. A 1.2-kb PstI-EcoRI fragment from
pMR105, containing the 5'-end of cheC and a cat
cassette (12), was cloned into PstI-EcoRI sites
of pAIN500 utilizing the EcoRI site internal to
cheD. The resulting plasmid (pAIN501) contained a
cat cassette flanked by the 5'-end of cheC and
the 3'-end of cheD.
Chromosomal DNA from strain PS29 (44) was used to transform a
cheB mutant (OI2715) (5) to spectinomycin resistance at a
locus immediately upstream of the fla/che operon to create
strain OI3349. Linearized pAIN501 was used to transform OI3349 cells. CmRSpcR colonies were selected, and the
resulting strain was called OI3375 (cheBCD). The generalized
transducing phage PBS1 was grown on OI3375 cells, and the resulting
lysate was used to transduce a cheR3::cat strain
(OI2680) (45), as described previously (46). The resulting
cheRBCD mutant was designated strain OI3377
(SpcRCmR). OI3377 SpcR
transductants were then back-crossed into strain OI1085 to confirm linkage between the spectinomycin marker and the cat
cassette located in the fla/che operon. In an independent
assay, the cotransduction frequency between the SpcR marker
and the fla/che operon was determined to be 94%.
Pulse Label Methylation--
In vivo pulse label
methylations were performed as described by Ullah and Ordal (39), with
the following changes: cells were grown to 180 Klett units (red filter)
in LB, washed three times in CB with 250 µg/ml chloramphenicol, and
resuspended at an A600 = 1.0 in the appropriate
volume of either CB or protoplast buffer with chloramphenicol. The
suspension was then incubated at 37 °C with moderate shaking (120 rpm). Methylation was initiated by addition of 50 µCi/ml
[3H]methionine (0.6 µM). At the appropriate
times, 1.0-ml samples were removed from flasks and frozen in a dry
ice/acetone bath. Samples were then thawed and centrifuged at 3000 × g for 30 min at 4 °C. The supernatant was either
assayed for volatile labeled product ([3H]methanol) or
discarded, and the protoplast membranes were resuspended in 100 µl of
4× SDS solubilizer. The samples were then boiled for 7 min and
electrophoresed at 15 V/cm on 10% SDS-polyacrylamide gels, pH 8.8 (47). The gels were then treated for fluorography as described
previously (48).
Steady-state Methylation--
For in vivo
steady-state methylation reactions, cells were treated as above except
that methylation was initiated with 50 µCi/ml of 10 µM
[3H]methionine. At the indicated times, 1.0-ml aliquots
were frozen in dry ice/acetone baths. For assays requiring the removal
of attractant, whole cells were washed by filtration using
0.45-µm-pore cellulose-acetate filters. The cells were resuspended in
CB at volumes equal to those before filtration and frozen in dry
ice/acetone at the times indicated. After thawing at 4 °C, an equal
volume of 2× protoplast buffer with 5 mg/ml lysozyme was added to each tube, and the tubes were incubated for 1 h at 4 °C. The
resulting protoplasts were then centrifuged and treated as described above.
Detection of Methanol from Protoplasts--
Volatile
radiolabeled product was detected using a continuous flow assay based
on the method described by Kehry et al. (32) and Thoelke
et al. (33). Protoplasts were labeled in vivo as described above for 15 min. 1.0-ml aliquots were frozen in dry ice/acetone baths at the times indicated. After thawing at 4 °C, the
protoplasts were pelleted by centrifugation, and 400-µl aliquots of
the supernatants were placed in 0.5-ml microcentrifuge tubes (without
lids). The microcentrifuge tubes containing the supernatants were then
placed in scintillation vials containing 2 ml of Liquiscint mixture
(National Diagnostics), allowed to equilibrate for 24 h, and counted.
Continuous Flow Assay for Methanol Production--
An assay
based on that described by Kehry et al. (32) has been
described previously (33). Cells were methylated as described above.
Labeled cells were loaded onto a 0.45-µm-pore cellulose-acetate filter and had CB pumped past them at a constant rate of 15 ml/min. The
buffer contained excess methionine (10 µM). When the
buffer reservoirs were changed, the cells experienced addition and
removal of the effector. The effluent was fractionated and assayed for volatile labeled product ([3H]methanol). A 400-µl
aliquot of each fraction was placed in a 0.5-ml microcentrifuge tube
(without a lid), and the microcentrifuge tubes were then placed in
scintillation vials containing 2 ml of scintillation fluid, allowed to
equilibrate for 24 h, and counted in a Beckman LS1701
scintillation counter.
Quantitation of Labeled Membrane Proteins--
The
3H-labeled proteins were visualized by fluorography (48),
and the entire region containing [3H]McpB was quantified
in units of absorbance with a PDI 420oe scanning densitometer with 42 µm resolution, using Quantity One® software.
Tethered Cell Assay--
The strain to be analyzed was grown
from a single colony without shaking in 1.0 ml of Tryptone broth. After
overnight growth, the culture was pelleted and resuspended in 250 µl
of Tryptone broth. The entire suspension was used to inoculate 25 ml of
minimal medium, and the culture was grown for 4.5 h at 37 °C
with vigorous shaking (250 rpm). Fifteen min prior to harvesting, 250 µl of a 5% glycerol/0.5 M sodium lactate solution was
added to the culture. The cells were placed in a 300-ml prechilled
Waring blender for 10 min at 4 °C. In order to shear the majority of
flagella, the cells were blended twice for 15 s, with a 45-s
intervening pause to allow cooling. A 250-µl aliquot of the blended
cells was then placed on a glass coverslip preincubated with 25 µl of
anti-flagellin antibody and incubated at room temperature for 30-45
min. The coverslip was then inverted and placed within a laminar flow
chamber to be observed by phase-contrast microscopy in a system similar to those described previously (5, 49). Chemotaxis buffer with or
without attractant was pumped through the laminar flow chamber at a
constant rate such that consumption of an attractant did not
significantly affect the concentration of the attractant. Rotating
cells were videotaped and analyzed by a Hobson Tracker, Bacterial
Edition (Hobson Tracking Systems Ltd., Sheffield, United Kingdom),
which generates text files containing continuous-time rotational data.
The data were processed by programs written with Matlab software (The
Mathworks, Inc.). For each cell, the continuous-time rotational data
was converted to discrete-time data with a step size of 0.1 s. All
the data points within a 4-s window were then averaged to generate a
data set with a step size of 4 s that contained the probability of
CCW rotation (smooth swimming behavior). The data for all cells in the
sample population were then pooled and averaged, and the resulting data
were smoothed over a five data point window to generate the final graph.
Swarm Plate Assay for Chemotaxis--
Strains were grown
overnight on tryptose blood agar base plates with the appropriate drug.
A single colony was transferred to a Tryptone semi-solid agar plate
(1% Tryptone, 0.5% NaCl, 1× Spizizen salts, 5 µg/ml required amino
acids, and 0.27% agar) and incubated at 37 °C for 4 h
(38).
Relationship between McpB Demethylation and Methanol
Production--
Previous experiments indicated that addition of the
attractant aspartate to wild-type B. subtilis (OI1085)
caused an immediate (within 24 s) loss of labeled methyl groups
from the MCPs under pulse-chase conditions (34). Under the same
experimental conditions, methanol production from OI1085 cells in
response to aspartate stimulation was observed to reach a maximum after
60 s (34). Those results, however, were obtained from analysis of
separate trials, and direct comparison of those results may not have
been reliable. Nevertheless, the apparent delay in methanol production relative to methylation changes on the MCPs supported the hypothesis that methyl groups were transferred to a stable intermediate methyl carrier from which methanol was subsequently released (29, 33). That
mechanism was proposed to account for the production of methanol that
has been observed in response to both positive and negative stimulation
for all amino acids in B. subtilis (28-30).
Recent experiments have shown that B. subtilis McpB is
required for methanol production in response to addition or removal of
asparagine (28). If methyl transfer occurs upon asparagine stimulation,
then a delay between McpB demethylation and methanol production might
be apparent. However, if no methyl transfer occurs, or if methyl
transfer is rapid relative to methanol release, then methanol
production and demethylation of McpB would appear to be coincident when
asparagine is added to the cells. In order to examine the relationship
between methanol production and demethylation of McpB, a time course of
methanol production and McpB demethylation was examined in an
McpB-expressing strain (OI3184) that lacks McpA, TlpA, and TlpB.
An in vivo pulse label assay was performed such that
[3H]methanol produced by the labeled OI3184 protoplasts
would be released into the surrounding medium and frozen at the various
time points of the experiment. After thawing the aliquots at 4 °C,
the supernatant was assayed for (volatile) [3H]methanol
(see under "Experimental Procedures"). The OI3184 protoplast membranes from those samples were fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by fluorography to detect changes in
McpB methylation. The results show that methanol production increased
dramatically within 5 s of addition of 0.5 mM
asparagine (calculated to be enough asparagine to titrate 90% of
receptors based on the experimentally determined KD;
50) (Fig. 1A). Demethylation
of McpB also occurred within 5 s of asparagine addition during
this assay (Fig. 1B). These data demonstrate that there is
no significant delay in methanol production following addition of
asparagine, in contrast to what was previously reported for aspartate
(34). Although the results of this experiment do not eliminate the
possibility of rapid methyl transfer to an intermediate carrier, no
methylated intermediate is necessary to account for the data in this
assay. Thus, it seems likely that methyl groups removed from McpB upon
asparagine addition are released directly as methanol. This methanol
production can therefore be attributed to the action of the CheB
methylesterase (4, 5), which is activated by CheA-P (2, 51) and is
similar to the process seen in E. coli in response to
repellent addition (31, 32).
Effect of Addition of Asparagine on McpB Methylation--
Previous
studies on the B. subtilis OI1085 strain (wild-type for
chemotaxis) also indicated that the attractant aspartate caused
increased turnover of methyl groups on the MCPs and that net
methylation changes did not occur (34, 37). In conjunction with that
finding, a return of 3H label to the MCPs after removal of
the aspartate under pulse-chase conditions was interpreted to mean that
reversible methyl transfer occurred during chemotaxis in B. subtilis (29). In order to test the methyl transfer hypothesis
specifically for McpB, a strain was constructed that lacked chromosomal
copies of mcpA, mcpB, mcpC,
tlpA, and tlpB at their normal loci and had
mcpB cloned into the amyE locus (strain OI3290).
A strain lacking mcpA, mcpB, mcpC,
tlpA, tlpB, and tlpC (OI3281; 42) has
no radiolabeled bands at the position where [3H]McpB
migrates in strain OI3290.5 TlpC migrates much
faster during SDS-polyacrylamide gel electrophoresis than does McpB
(52), and it therefore does not interfere with the analysis of McpB in
strain OI3290. Thus, in strain OI3290 it is possible to follow
methylation changes specifically in McpB that occur in response to
asparagine stimulation.
OI3290 cells were labeled with 10 µM
[3H]methionine. The methylation reached a steady-state
level by 30 min (Fig. 2A).
Addition of 0.5 mM asparagine induced rapid net
demethylation of McpB. The first time point was taken 5 s after
addition of asparagine, by which time a net change in methylation had
already taken place. A decrease of approximately 50% in the total
level of methylation prior to stimulation occurred and was accompanied
by the appearance of a more slowly migrating, presumably less
methylated, species (Fig. 2B). The rapid net demethylation
was similar to that observed in response to repellent stimulation in
E. coli (53). Following this initial demethylation of McpB,
however, both methylated species were gradually remethylated such that
the total level of methylation returned to near prestimulus levels.
This remethylation occurred even in the continued presence of the
attractant. By contrast, in E. coli, the net demethylation
due to repellent addition persists for at least 30 min (53). Because
the data support the hypothesis that methyl groups are released
directly as methanol (Fig. 1), the poststimulus remethylation of McpB
could be catalyzed by the CheR methyltransferase using AdoMet as a
substrate (7).
Effect of Removal of Asparagine on McpB Methylation--
Addition
and removal of attractants and repellents have opposite effects on the
behavior of both B. subtilis and E. coli in tethered cell assays (5, 45, 54). Furthermore, positive and negative
stimuli have opposite effects on the final methylation state of the
receptors in E. coli (53). Therefore, we hypothesized that
removal of asparagine might lead to a transient increase in
the overall methylation level of McpB followed by a gradual decrease in
the methylation state of the receptor.
However, it is apparent that the same effect occurs upon removal of
attractant as upon addition of attractant (Fig. 2A). Both addition and removal of 0.5 mM asparagine induced rapid net
demethylation of McpB. The first time point taken after removal of
asparagine was 30 s, by which time demethylation was already
complete. Again, the extent of demethylation was approximately 50%.
After this rapid demethylation, gradual remethylation of McpB occurred
over a period of several minutes, as indicated by the disappearance of
the more slowly migrating species (Fig. 2C).
To verify that the apparent changes in methylation of McpB are due to
net methylation changes and not a net loss of protein, Western blots
were performed on the samples over the time course of the experiment
shown in Fig. 2. The results indicate that both methylated species
cross-react with anti-McpB antibody and that the total amount of
protein was the same before and after addition of attractant (data not
shown). Anti-McpB antibody does not cross-react with any other protein
that comigrates with McpB under the conditions tested.6 Thus,
all methylation changes on McpB can be attributed to net methylation of
McpB and not to any loss of protein.
Together, these results provide an explanation for the previous
observations that methanol is produced by B. subtilis in
response to all stimuli and that the MCPs are relabeled when attractant stimuli are removed (29). No reversible methyl transfer need be
postulated to occur during chemotaxis in B. subtilis, in
contrast to what was previously hypothesized.
Effect of a cheY Mutation on Methylation of McpB--
Recently, it
has been shown that methanol production upon removal of asparagine in
the continuous flow assay requires the response regulator CheY (28).
Because methanol production after removal of attractant can be
attributed to a second demethylation event, we hypothesized that a
cheY knockout mutation would prevent demethylation of McpB
caused by removal of asparagine. A derivative (strain OI3294) of strain
OI3290 containing a deletion in the cheY gene was made to
test this possibility. The cheY mutation did not
significantly alter the extent of initial demethylation of McpB after
asparagine addition (Fig. 3). However,
remethylation of McpB did not occur when CheY was absent. After removal
of asparagine, however, remethylation of McpB occurred over a time
course similar to that of strain OI3290 (Figs. 2 and 3). The second
demethylation event, which produces methanol when the attractant is
removed (Fig. 2), cannot occur in the cheY mutant because
remethylation of asparagine-bound McpB does not occur. It should be
noted that this behavior in the B. subtilis cheY mutant is
similar to that seen in wild-type E. coli upon repellent
addition and removal (54). It is also reciprocally related to the
results obtained in E. coli upon attractant addition and
subsequent removal (53, 54).
Close inspection of the time course of methylation in the
cheY mutant indicated that maximal demethylation of McpB was
not reached until several minutes after asparagine was added (compare Figs. 2A and 3A). Likewise, the more slowly
migrating, presumably less methylated species of McpB only gradually
became apparent in strain OI3294, in contrast to the immediate
formation of that band in strain OI3290 (compare Figs. 2B
and 3B). Thus, CheY accelerates overall methylation changes
on McpB by an unknown mechanism.
Effect of the cheY54DA Mutation on Methanol
Production--
Although CheY is clearly required for the
remethylation of asparagine-bound McpB, the assay described above did
not differentiate between a requirement for the phosphorylated or
unphosphorylated form of CheY. In order to determine which form of CheY
is required, a strain (OI2952) (3) containing a point mutation
(cheY54DA) that renders CheY incapable of being
phosphorylated was assayed for its ability to produce methanol. Because
methanol production upon asparagine addition and removal can be
attributed to the demethylation of McpB, the continuous flow assay for
methanol production can be used to track both the demethylation and
remethylation events. Strain OI2952 was capable of producing methanol
upon addition of asparagine but could not produce methanol when the
asparagine was removed (Fig.
4A). The response by strain
OI1085 (wild-type for chemotaxis) is shown for comparison (Fig.
4B). Presumably, the alanine substitution simply prevents
the mutant CheY54DA protein from becoming phosphorylated and does not
create any other significant conformational defects. Therefore, the
result of this assay allows us to conclude that the remethylation of
asparagine-bound McpB (Fig. 2) specifically requires CheY-P.
Behavior of a cheRBCD Mutant--
B. subtilis proteins
thought to be involved in adaptation include the methyltransferase
(CheR), the methylesterase (CheB), CheC, and CheD (5, 12, 13, 45). The
results described above suggest that CheY-P may interact with
asparagine-bound McpB, either directly or indirectly. Increased levels
of CheY-P after asparagine stimulation could affect the conformation of
the signaling complex to lower the level of CheA autophosphorylation,
thereby promoting adaptation. To test this hypothesis, a
cheRBCD mutant was constructed for behavioral analysis. If
CheY-P does not facilitate adaptation and adaptive methylation no
longer exists, then the cheRBCD mutant should become 100%
CCW (smooth swimming) upon asparagine stimulation and remain smooth
swimming. However, if CheY-P can promote adaptation independently of
changes in methylation, then we would observe a diminution of CCW
flagellar bias after the initial excitation. The results show that the
cheRBCD mutant excites transiently and undergoes subsequent
oscillations (Fig. 5A). In contrast, wild-type cells (OI1085), after transiently increasing their
CCW bias in response to attractant stimulation, return to their
prestimulus bias (about 60% CCW) within 60 s and maintain that
bias in the presence of the attractant (Fig. 5B) (3, 5). It
should be noted that cheY mutant cells (OI2057) are
completely tumbly (0% CCW) and incapable of increasing their CCW
flagellar bias in response to attractant stimulation (3). Thus, the
cheRBCD mutant cells, although capable of responding to
attractant stimuli, do not return to their prestimulus CCW bias nor
maintain a stable, adapted state in the presence of an attractant.
After the asparagine is removed from the cheRBCD mutant
cells, the oscillation immediately ceases. Why the poststimulus CCW
bias of the mutant remains low relative to its prestimulus level is
unknown. However, recent evidence indicates that the flagellar bias of
the cheRBCD mutant gradually returns to its prestimulus
level over a period of 10-15 min following removal of
asparagine.6
Previous behavioral analysis of Salmonella typhimurium cells
by Spudich and Koshland (55) demonstrated that cells within a
population exhibit considerable individualistic variation. Likewise, our study found that there is considerable heterogeneity among all
cells analyzed in the tethered cell assay. Overall, two-thirds of the
cheRBCD mutant cells show the oscillating phenotype, and one-third give partial adaptation. These results are highly
reproducible. For Fig. 5A three cells were averaged, all
within one visual field of our tracking device. Other fields showed
similar results. The cells within any given visual field were observed
to oscillate synchronously with a relatively long time constant.
However, because the exact time when attractant reaches the cells for
each field is not known, summation of several fields was not possible
without introducing phase variation that smoothes the data. Thus, the fact that a number of these B. subtilis mutant cells show
synchronized oscillations implies that a feedback system not requiring
CheR, CheB, CheC, or CheD exists. Although other proteins involved in adaptation may exist, these results suggest that the decrease in CCW
rotation that follows the initial excitation may be due to CheY-P
feedback that results in lowering the rate of CheA autophosphorylation. As a result, CheY-P levels would decrease and feedback inhibition would
cease. Because asparagine is still bound to McpB, CheA
autophosphorylation would then increase to produce increased levels of
CheY-P, allowing the cycle to repeat, thereby producing the observed
oscillation (Fig. 5A).
Swarm Plate Analysis for Chemotaxis by the cheRBCD Mutant--
In
order to test the efficiency of the putative CheY-P feedback mechanism,
the cheRBCD mutant was analyzed for its ability to swarm in
a Tryptone swarm plate. The results show that the cheY
mutant (OI2057) is unable to produce a swarm in Tryptone semi-solid
agar, whereas both the wild-type (OI1085) and the cheRBCD mutant (OI3377) strains are capable of swarming in a Tryptone swarm
plate (Fig. 6). Although the possibility
remains that adaptation may not be required to produce effective
swarming under the conditions tested, we do not believe such a well
defined ring, the same size as wild-type, could form without a
mechanism for adaptation by the cheRBCD mutant. Furthermore,
the swarm cannot be due to oxygen taxis because the swarm diameter is
equal through the depth of the agar. Oxygen taxis rings are
characteristically larger at the base of the swarm under the conditions
tested (56). Thus, we conclude that an adaptational mechanism exists in
the cheRBCD mutant that facilitates chemotactic swarming by
B. subtilis and that this mechanism may involve feedback of
CheY-P onto asparagine-bound McpB. The exact mechanism by which
adaptation would be produced remains unknown.
In this study, we produce evidence that methanol is released
directly from the MCPs (Fig. 1), a process known to be mediated through
the action of the methylesterase, CheB-P (4, 5). We also show that both
addition and removal of asparagine result in transient net
demethylation of McpB (Fig. 2). Following both demethylation events,
McpB is remethylated (Fig. 2) presumably by CheR using AdoMet as a
substrate (7). Together, these results provide an explanation for the
previous observations that the MCPs are relabeled upon removal of
aspartate and methanol production occurs for both addition and removal
of aspartate (29). Thus, no methyl transfer is necessary to account for
the observations reported in this study or the previous findings of
Thoelke et al. (29). Because it has recently been shown that
all 20 amino acid attractants for B. subtilis induce
methanol production in the continuous flow assay both upon their
addition and removal (28), it is likely that all 20 amino acids induce
net demethylation upon binding and release from their receptors. Each
demethylation event should be followed by remethylation of the receptor.
We also show that the inability of the cheY null mutant to
remethylate asparagine-bound McpB (Fig. 3) accounts for the lack of
methanol production in response to asparagine removal from the
cheY mutant during the continuous flow assay (28). Methanol production after asparagine removal is also inhibited in cells containing the unphosphorylatable CheY54DA mutant protein (Fig. 4).
These results allow us to conclude that remethylation of
asparagine-bound McpB requires CheY-P, leading to the hypothesis that
CheY-P may participate in a feedback mechanism that promotes adaptation
to positive stimuli during chemotaxis in B. subtilis. This
hypothesis is supported by the observations that the cheRBCD
mutant exhibits transient excitation and subsequent oscillation in the
tethered cell assay and that the cheRBCD mutant is capable
of swarming in a Tryptone swarm plate.
The cheRBCD mutant lacks all proteins thought to be involved
in adaptation during chemotaxis in B. subtilis (5, 12, 13, 45). Although the possibility remains that other chemotaxis proteins
involved in adaptation may exist, there is no evidence to suggest that
is the case. No sequence similar to E. coli CheZ has been
found in a search of the B. subtilis genome data base in
GenBankTM. Thus, the ability of the majority of the cheRBCD mutant cells to exhibit transient excitation in an oscillating fashion
supports the hypothesis that CheY-P may interact with asparagine-bound
McpB to inhibit CheA autophosphorylation. If McpB possesses CheZ-like
activity, it might influence the dephosphorylation of CheY-P, and the
system could be "re-excited," as was observed (Fig. 5A).
How this potential feedback mechanism would allow the cheRBCD mutant to swarm in a Tryptone swarm plate is unknown
(Fig. 6). Furthermore, the nutrient(s) upon which the cells feed and toward which they subsequently migrate in Tryptone swarm plates is
unknown, and thus the specific receptor or group of receptors mediating
this behavior is also unknown. Nevertheless, it is apparent that the
cheRBCD mutant possesses a mechanism that allows for efficient swarming (Fig. 6). One possibility is that the oscillations observed in the tethered cell assay may not occur for the
cheRBCD cells in Tryptone swarm plates, and thus, a putative
feedback mechanism may result in partial adaptation that allows for
chemotactic swarming.
In strain OI3290, McpB released methanol upon asparagine stimulation,
and the protein immediately appeared as a more slowly migrating, less
methylated species (Fig. 2B). In contrast, in the
cheY mutant, a more slowly migrating, less methylated
species became apparent only several minutes after asparagine
stimulation (Fig. 3B). If in the cheY mutant,
CheB were phosphorylated to a greater extent, then the more slowly
migrating, less methylated form of McpB would be expected to appear
more rapidly. Because the band emerged less rapidly instead, we
conclude that CheY-P interaction with asparagine-bound McpB facilitates
a conformational change influencing these methylation changes in
addition to lowering the CheA autophosphorylation rate. The possibility
remains, however, that the inability to remethylate the ligand-bound
receptor in the cheY mutant (Fig. 3B) may be due
to a relative increase in CheB-P levels, thereby preventing a net
increase in methylation by CheR. Nevertheless, we speculate that these
methylation changes are required for generating the adapted state in
the wild-type strain (Fig. 5B), a state that is not
generated in the cheRBCD mutant (Fig. 5A).
How could CheY-P feedback influence both CheA autophosphorylation and
methylation of asparagine-bound McpB? Methylation of chemotactic
receptors in E. coli is thought to affect the interactions between the methylated helices of the MCPs such that CheA activation is
enhanced by a more highly methylated receptor (57-62). Because a
B. subtilis cheR mutant has a lower prestimulus flagellar
rotational bias (reflecting lower CheA activity), we conclude that
enhanced CheA activity results from increased methylation for B. subtilis (45). Although enhanced CheA activity leads to tumbling
in E. coli (22-27) and smooth swimming in B. subtilis (2, 3), it appears that enhanced CheA activity results
from increased methylation for both organisms. In B. subtilis, we speculate that CheY-P interaction with
asparagine-bound McpB could affect the topology of the C terminus of
the receptor such that CheB-P has access to an otherwise less
accessible methylated residue (Fig. 7).
The demethylated, asparagine-bound receptor would presumably promote a
lower rate of CheA autophosphorylation, thereby generating an adapted
state.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B. subtilis strains used in this study
Plasmids and phage used in this study
) at the BamHI-XbaI sites to
create pUNK101. Subsequently, a 2.8-kb SmaI-XbaI
fragment containing mcpB from pUNK101 was subcloned into the
SmaI-XbaI sites of pHL007, which is a derivative
of the amyE integration plasmid, pAC7 (40), and contains a
kanamycin resistance
marker.3 The
resulting plasmid (pUNK200) carries mcpB under the control of its natural promoter.
) were
shown to express only one band that cross-reacts with anti-McpB antibody,5 and it
produces only one methylatable protein that migrates to the expected
position in a 10% SDS-polyacrylamide gel, based on the
mcpB1::cat phenotype observed by Hanlon and Ordal
(1).
) were then
transformed with chromosomal DNA from strain OI3280 (mcpC4::erm; 42). EmR transformants
were selected, and the resulting strain (OI3294) was tested to verify
that it produces only one methylated protein at the position to which
McpB is expected to migrate, as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Time course of methanol evolution and net
demethylation of McpB in response to asparagine stimulation. The
assay was performed as described under "Experimental Procedures."
The arrow indicates the time of addition of 0.5 mM asparagine to strain OI3184. A, methanol
production. B, net demethylation of MCPs. The data represent
the average of three trials. Error bars represent the
S.D.
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Fig. 2.
Methylation changes on McpB in response to
addition and removal of asparagine. The assay was performed as
described under "Experimental Procedures." A, The
downwardly directed arrow indicates the time of addition of
0.5 mM asparagine to strain OI3290. The upwardly
directed arrow indicates the time of removal of same. The data
represent the average of three trials. Error bars represent
the S.D. B, fluorograph of [3H]McpB showing
methylation changes in response to addition of asparagine. Lanes
1 and 2 were sampled 40 min after addition of
[3H]methionine and prior to addition of asparagine.
Following addition of asparagine, samples were taken at 15 s
(lanes 3 and 4), 2 min (lanes 5 and
6), 4 min (lanes 7 and 8), 8 min
(lanes 9 and 10), and 16 min (lanes 11 and 12). C, fluorograph of [3H]McpB
showing methylation changes due to removal of asparagine. Lane
1, 16 min after addition of asparagine and prior to removal of
asparagine (as in B, lanes 11 and 12). Following
removal of asparagine, samples were taken at 35 s and 1, 2, 4, 8, 16, and 24 min (lanes 2-8, respectively).
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Fig. 3.
Methylation changes in McpB in a
cheY mutant in response to addition and removal of
asparagine. The assay was performed with strain OI3294 as
described under "Experimental Procedures." A, the
downwardly directed arrow indicates the time of addition of
0.5 mM asparagine to strain OI3294. The upwardly
directed arrow indicates the time of removal of same. The data
represent the average of three trials. Error bars represent
the S.D. B, fluorograph of [3H]McpB showing
methylation changes following addition of asparagine. Lanes
1 and 2 were loaded with samples taken 40 and 50 min,
respectively, after addition of [3H]methionine and prior
to addition of asparagine. Lanes 3-8 were loaded with
samples taken 30 s and 1, 2, 4, 8, and 16 min after addition of
asparagine, respectively.
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Fig. 4.
Methanol production by
cheYD54A mutant and wild-type cells following
stimulation with asparagine. The assay was performed as described
under "Experimental Procedures." The downwardly directed
arrow indicates the time of addition of 0.5 mM
asparagine. The upwardly directed arrow indicates the time
of removal of same. A, response by the cheY54DA
mutant (strain OI2952). B, response by the wild-type (strain
OI1085).
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Fig. 5.
Behavior of cheRBCD and
wild-type tethered cells in response to asparagine stimulation.
The assay was performed as described under "Experimental
Procedures." The downwardly directed arrow indicates the
time of addition of 56 µM asparagine. The upwardly
directed arrow indicates the time of removal of same.
A, the rotational data for 3 cheRBCD mutant cells
(strain OI3377) from one visual field were averaged to generate the
plot shown. Phase variation between fields prevented summation of
several fields. B, the rotational data for 15 wild-type
cells (strain OI1085) were averaged to generate the plot shown.
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Fig. 6.
Swarm plate assay of a cheRBCD
mutant. The assay was performed as described under
"Experimental Procedures." The plate was inoculated with
(clockwise from top) strains OI1085 (wild-type), OI2057
( cheY), and OI3377 (cheRBCD). The Tryptone
swarm plate was incubated at 37 °C for 4 h.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Model for CheY-P feedback on asparagine-bound
McpB. This model depicts events leading to an adapted state for
ligand-bound McpB. Prior to stimulation, McpB is methylated (Me) at one
or more of several sites (solid black lines). Following
addition of asparagine, the receptor complex is active, leading to
increased production of CheY-P and, therefore, smooth swimming behavior
in B. subtilis. Feedback by CheY-P onto the receptor
(orange dashed arrow) is thought to alter the conformation
of the ligand-bound receptor and results in the following: 1) the rate
of CheA autophosphorylation is transiently lowered, thereby promoting
adaptation to a positive stimulus, and 2) CheB-P gains accessibility to
a methylated residue (black triangle), enhancing
demethylation at that site. In the cheY mutant,
demethylation of this residue occurs more slowly, as indicated by the
more gradual appearance of the more slowly migrating form of McpB (Fig.
3). Furthermore, in the absence of methylation changes in the
cheRBCD mutant, oscillation between the active and
transiently inactive states would occur due to spontaneous or
receptor-enhanced hydrolysis of CheY-P. The brackets around
the active and transiently inactive states indicate that CheB-P can
remove methyl groups from either form of the receptor complex.
Demethylation of ligand-bound McpB would presumably create a stable
adapted state, a prerequisite for chemotaxis. Methylation of
ligand-bound McpB by CheR does not destroy the adapted state, as
indicated by the observation that this event occurs well after
behavioral adaptation has occurred. This methylation may occur at a
unique site and play a role in presetting the complex to respond to the
removal of the attractant (see Footnote 5).
The data from this study allow us to account for the unusual changes in
methylation of the MCPs and methanol production previously observed in
B. subtilis (29). Feedback by CheY-P onto asparagine-bound McpB would provide a simple mechanism to regulate the activity of the
sensor kinase, CheA, while also affecting methylation in an unknown
way. If CheY-P interacts with McpB, this would be the first example of
a response regulator that has two targets (Fig. 7): one upstream of the
sensor kinase (asparagine-bound McpB) and one downstream (the flagellar
switch) (see Ref. 63 and references therein). Because the archael
species Halobacterium salinarium, like B. subtilis, produces methanol in response to all stimuli (28, 64),
it is possible that CheY-P feedback onto the receptors was part of an
adaptation mechanism present in a common ancestor of these two highly
diverged prokaryotic organisms.
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ACKNOWLEDGEMENTS |
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We thank Bob Bourret for valuable discussions and critical analysis and reading of the manuscript. We also thank Shelby Feinberg for performing the continuous flow assay on the cheY54DA mutant. We thank A. L. Sonenshein for the donation of B. subtilis strain PS29. J. R. Kirby thanks Maqsudul Alam, Gerald Hazelbauer, Stan Maloy, Bastianella Perazzona, John Spudich, Jeff Stock, Barry Taylor, Alan Wolfe, and Igor Zhulin for valuable discussions.
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FOOTNOTES |
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* Support for this research was provided by Public Health Service Grants AI20336 and GM54365 from the National Institutes of Health (to G. W. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Molecular and Cell Biology, University
of California, Berkeley, CA 94720.
¶ To whom correspondence should be addressed: 190 Medical Science Bldg., 506 S. Mathews, University of Illinois, Urbana, IL 61801. Tel.: 217-333-9098; Fax: 217-333-8868; E-mail: g-ordal{at}uiuc.edu.
2 Garrity and Ordal, unpublished data.
3 H. Lu and G. W. Ordal, unpublished data.
4 M. M. L. Rosario and G. W. Ordal, unpublished data.
5 Kirby, J. R., Niewold, T. B., and Ordal, G. W., submitted for publication.
6 M. M. Saulmon and G. W. Ordal, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: CCW, counterclockwise; AdoMet, S-adenosylmethionine; cat, chloramphenicol acetyltransferase; MCP, methyl-accepting chemotaxis protein; Tlp, transducer-like protein; CB, chemotaxis buffer; bp, base pair(s); kb, kilobase pair(s).
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