ABSTRACT
CheZ is the phosphatase of CheY, the response regulator in
bacterial chemotaxis. The mechanism by which the activity of CheZ is
regulated is not known. We used cheZ mutants of Salmonella
typhimurium, which had been isolated by Sockett et al. (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V. M., and
Macnab, R. M.(1992) J. Bacteriol. 174, 793-806), for
cloning the mutant cheZ genes, overexpressing and purifying
their products. We then measured the phosphatase activity, binding to
CheY and to phosphorylated CheY (CheY
P), and CheY
P-dependent
oligomerization of the mutant CheZ proteins. While all the mutant
proteins were defective in their phosphatase activity, they bound to
CheY and CheY
P as well as wild-type CheZ. However, unlike
wild-type CheZ, all the four mutant proteins failed to oligomerize upon
interaction with CheY
P. On the basis of these and earlier results
it is suggested that (i) oligomerization is required for the
phosphatase activity of CheZ, (ii) the region defined by residues
141-145 plays an important role in mediating CheZ oligomerization
and CheY
P dephosphorylation but is not necessary for the binding
to CheY
P, (iii) the oligomerization and hence the phosphatase
activity are regulated by the level of CheY
P, and (iv) this
regulation plays a role in the adaptation to chemotactic stimuli.
Regulation of bacterial chemotaxis is essentially regulation of
the direction of flagellar rotation(1) . This is done by
phosphotransfer reactions ending up with the phosphorylation of CheY,
the signal protein in bacterial chemotaxis (for recent reviews see, (2, 3, 4, 5, 6) ).
Phosphorylated CheY (CheY
P) (
)interacts with the switch
at the base of the flagellar motor(7, 8, 9, 10, 11, 12) (see
Refs. 6 and 13 for recent reviews on the switch) with a resultant
clockwise (CW) rotation(14) . (The default direction of
rotation is counterclockwise
(CCW)(8, 9, 15, 16, 17) .)
CheY is phosphorylated by an autophosphorylatable kinase,
CheA(18, 19, 20) . This activity is regulated
by chemotactic stimuli(21, 22, 23) .
CheY
P is dephosphorylated by CheZ (19, 20) .
However, unlike the kinase, no regulation of the phosphatase activity
of CheZ has been found, even though a computer simulation of signal
transduction (24) implicated the occurrence of such a
regulation mechanism.
In addition to its phosphatase activity, CheZ
was found to be involved in two other processes: binding to CheY (25, 26) (mainly to its phosphorylated
form(26) ), and oligomerization upon binding to CheY
P (27) . With the goal of finding out whether the oligomerization
is involved in CheZ regulation, this study examines, by biochemical
analysis of mutant CheZ proteins, the inter-relationship between the
different functions of CheZ.
EXPERIMENTAL PROCEDURES
Bacterial Strains
The strains used in this study
are listed in Table 1.
Cloning of Mutant cheZ Genes onto an Overexpressing
Vector
For the overexpression of mutant CheZ proteins we used a
collection of cheZ mutants generated and characterized by
Sockett et al.(28) . The cheZ mutant alleles
were amplified from total DNA of the respective Salmonella
typhimurium strain by polymerase chain reaction using the primers
5`-CCGAATTCATGATGCAACCATCTATCAAGCC-3` and
5`-CCGGATCCTTAACAGCCAAGACTGTCCAGCA-3`, which contained added EcoRI and BamHI sites, respectively, at their 5` end.
The amplified cheZ-containing fragments were digested with EcoRI and BamHI, and ligated with pBTac1 (Boehringer
Mannheim) predigested with EcoRI and BamHI (Fig. 1). The resultant plasmids (Table 2) overexpressed
the wild-type and mutant CheZ proteins under the control of the tac promoter. The existence of the mutations in the cloned cheZ genes and the lack of additional mutations that might be caused by
polymerase chain reaction were confirmed by DNA sequencing.
Figure 1:
Schematic diagram of the construction
of the CheZ-overexpressing plasmids.
Overexpression and Purification of CheZ
Wild-type
CheZ and the mutant proteins CheZ141FI and CheZ143DE were overexpressed
in RP3098. CheZ110LP was overexpressed in RP1616, and CheZ145TM in BW3.
The purification of CheZ was carried out as described in the preceding
paper (27) except that, in the case of CheZ110LP and CheZ145TM,
buffer A used for washing the Sepharose CL-6B column, contained 225
mM NaCl (instead of 275 mM), and the elution of CheZ
was carried out by a gradient of 225-450 mM NaCl
(instead of 275-450 mM). The overexpression and
purification of CheY from S. typhimurium was described in the
preceding paper (27) .
Sensitivity of CheZ to Proteolysis
CheZ (83
µM) in Tris-HCl (50 mM, pH 7.9) and MgCl
(5 mM) were incubated at room temperature (22 °C)
with trypsin (Sigma T-8642, 4 µg/ml). Samples of 10 µl were
removed at the indicated time points, quenched by addition of 3 µl
of
5 concentrated sample buffer and 10 min boiling, and analyzed
by 15% SDS-polyacrylamide gel electrophoresis.
Phosphatase Activity of CheZ
The phosphatase
activity of CheZ was assayed by monitoring the steady-state level of
CheY phosphorylation in the presence of
[
P]acetyl phosphate (AcP) as described
earlier(27) .
CheZ Radiolabeling
CheZ was radiolabeled by
methylating the
-amine of its lysine residues with formaldehyde
and NaB[
H]H
: a mixture (100 µl)
of CheZ (100 µM), H
BO
-NaOH (0.2 M, pH 9.0), formaldehyde (5 mM), and
NaB[
H]H
(170 µM, 24
Ci/mmol, obtained from Amersham) was incubated on ice for 25 min, and
then the reaction was terminated by the addition of 100 µl of
Tris-HCl (50 mM, pH 7.9). The radiolabeled CheZ
(1200-1500 cpm/pmol) was separated from the unreacted
NaB[
H]H
by a brief spin at 480
g in a 0.8-ml G-50 mini-column followed by dialysis
against Tris-HCl containing 0.2 mM phenylmethylsulfonyl
fluoride, and stored at -20 °C.
Binding of CheZ to CheY
Binding of CheZ to CheY
immobilized onto CNBr-activated Sepharose beads was measured as
follows. Immobilized CheY (50 µM) or immobilized bovine
serum albumin (as a control) were prepared as described(26) .
The reaction mixture (200 µl), consisting of CheY- or BSA beads,
Tris-HCl (50 mM, pH 7.9), MgCl
(7.7 mM),
glycerol (5.3%), bovine serum albumin (2.7 mg/ml),
[
H]CheZ (0.2 µM, 48,000-60,000
cpm), and, where indicated, AcP (18 mM), was incubated for 15
min at room temperature (22 °C). The beads were then pelleted by a
brief centrifugation, and washed twice by 0.5 ml of Tris-HCl (50
mM, pH 7.9), MgCl
(5.0 mM), and AcP (18
mM, only when it had been included also in the assay mixture).
The bound protein was extracted from the beads by agitating them in 400
µl of SDS (10%) for 1 h, after which the beads were pelleted and
the amount of [
H]CheZ in the supernatant was
determined by scintillation counting.
Cross-linking
Cross-linking of
[
H]CheZ (wild type and mutants,
30,000-36,000 cpm per each reaction mixture) was carried out by a
mixture of 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride
and N-hydroxysuccinimide as described in the preceding paper (27) .
RESULTS
Cloning of Mutant cheZ Genes, and Overexpression and
Purification of Their Products
In order to determine whether any
of the functions of CheZ (binding to CheY, phosphatase activity, and
oligomerization) are correlated, we chose to analyze these functions in
several mutant CheZ proteins. To this end we used S. typhimurium
cheZ point mutants isolated and characterized by Sockett et
al.(28) (Table 1). Flagellar rotation in all these
mutants is CW biased, indicating that these cheZ alleles code
for CheZ proteins with impaired ability to antagonize the CW causing
activity of CheY in vivo. To produce the proteins, we
amplified the mutant genes by polymerase chain reaction directly from
the chromosomal DNA of the mutant strains, and cloned the amplified
genes into the expression vector pBTac ( Fig. 1and Table 2). The plasmids containing the cloned genes indeed
overexpressed the mutant CheZ proteins, thus enabling us to purify the
proteins to near homogeneity (Fig. 2).
Figure 2:
Gel electrophoresis of the purified mutant
CheZ proteins. Each of the purified mutant CheZ proteins (5 µg)
were analyzed on a 15% SDS-polyacrylamide gel electrophoresis. Lanes 1-5 are wild-type CheZ, CheZ110LP, CheZ141FI,
CheZ143DE, and CheZ145TM, respectively. Lane 6 contains the
indicated molecular size markers (in kDa).
The Phosphatase Activity of Mutant CheZ
Proteins
The phosphatase activity of each of the mutant CheZ
proteins was determined by measuring its effect on the level of CheY
phosphorylation under steady-state conditions. All the mutant proteins
had a significantly lower phosphatase activity than wild-type CheZ (Fig. 3). For example, in the presence of 2 µM CheZ, the level of CheY
P under steady-state conditions was 7%
in the wild type versus 75, 76, 62, or 37% in CheZ110LP,
CheZ141FI, CheZ143DE, or CheZ145TM, respectively (100% is the
steady-state level of CheY
P in the absence of CheZ). This means
that CheZ110LP and CheZ141FI had over 10-fold lower phosphatase
activity than the wild type, and CheZ 143DE and CheZ145TM had 9- and
5-fold lower activities, respectively.
Figure 3:
Phosphatase activity of mutant CheZ
proteins. The activity is represented by the steady-state level of
CheY
P after 10 min incubation with varying concentrations of CheZ.
The fraction of CheY
P out of total CheY (0.26 ± 0.06) in
the absence of CheZ was considered as 100%. The results are the mean
± S.D. of three independent experiments. Closed circles, wild-type CheZ; open circles, CheZ110LP;
symbols,
CheZ141FI; squares, CheZ143DE; triangles,
CheZ145TM.
Binding of Mutant CheZ Proteins to CheY
The
apparent defects in the phosphatase activity of the mutant CheZ
proteins could result from one of the following possibilities: (i) a
reduced binding to CheY
P; (ii) an increased binding to
non-phosphorylated CheY, as a result of which the CheZ-CheY complex
could not dissociate and CheZ could not become available to other
CheY
P molecules (CheZ was used in catalytic concentrations); or
(iii) a reduced catalytic activity. To distinguish between these
possibilities, we measured the binding of the mutant CheZ proteins to
both phosphorylated and non-phosphorylated CheY. For this purpose the
binding of [
H]CheZ to CheY immobilized onto
CNBr-activated Sepharose beads was determined in the presence or
absence of the phosphodonor AcP(29) . We used a large excess of
CheY and AcP, thus making the loss of CheY
P, resulting from the
phosphatase activity, negligible. As shown in Fig. 4, all the
mutant CheZ proteins were bound to the CheY beads to an extent similar
to that of wild-type CheZ under both phosphorylating and
non-phosphorylating conditions. These results, obtained with
subsaturating concentrations of CheZ, indicate that the mutant CheZ
proteins are not defective in their binding to either CheY
P or
CheY. Thus, by way of elimination, it seems that the third possibility, i.e. a reduced catalytic activity, is the cause of the
apparent defects in phosphatase activity of the mutants.
Figure 4:
Binding of the mutant CheZ proteins to
phosphorylated and non-phosphorylated CheY. The results are the mean
± S.D. of four independent experiments. The binding level of
wild-type CheZ to CheY beads in the presence of AcP (19 ± 2% of
the amount of CheZ added) was considered as relative binding =
1. The hatched and black columns stand for the
absence and presence of AcP, respectively.
Oligomerization of the Mutant CheZ Proteins
As
before(27) , we used cross-linking to determine the occurrence
of CheZ oligomerization. Under non-phosphorylating conditions, i.e. in the absence of AcP, similar cross-linking products were
obtained with the wild-type and mutant proteins. Thus, in the absence
of CheY, the major band was that of the CheZ dimer (Fig. 5, lanes 2, 5, 8, 11, and 14; the molecular size of the
monomer is 23.9 kDa(30) ); in the presence of CheY but absence
of AcP, higher molecular size products (up to
70 kDa) were also
observed (lanes 3, 6, 9, 12, and 15). However, under
phosphorylating conditions there was a marked difference between the
wild type and the mutants: all the mutant proteins (lanes 7, 10,
13, and 16), unlike the wild-type protein (lane
4), did not form the high molecular size oligomer with CheY
P.
(In the case of CheZ110LP, other forms of CheZ, higher than the dimer
but lower than the CheZ-CheY
P oligomer, were also observed,
possibly because of its global structure defects (see below).) These
results demonstrate that the mutant CheZ proteins are unable to
oligomerize. When taken together with the conclusion reached earlier
that all the mutants have a reduced catalytic activity, the results
further suggest that the oligomerization is involved in the phosphatase
activity of CheZ.
Figure 5:
Cross-linking of wild-type and mutant
CheZ proteins. The figure contains autoradiograms of cross-linking
products resolved on a 10% polyacrylamide gel. The cross-linking was
carried out for 40 min as described under ``Experimental
Procedures.'' The high molecular size bands observed in the
presence of cross-linker only (lanes 2, 5, 8, 11 and 14) are the result of aggregates too large to enter the
resolving gel.
The Effect of the Mutations on the Global Structure of
CheZ
To determine whether the strong effects of the point
mutations on the oligomerization and the phosphatase activity of CheZ
were the consequence of local effects or global perturbation of CheZ
structure, we measured the susceptibility of the proteins to limited
trypsin proteolysis. The rationale behind this approach was that
perturbation in the structure of CheZ is expected to make the protein
less compact and thereby to expose more sites to trypsin action, with a
consequent faster proteolysis. As shown in Fig. 6, the mutant
proteins CheZ141FI, CheZ143DE, and CheZ145TM exhibited similar
proteolysis patterns as wild-type CheZ, indicative of no major
structural changes in these three mutant proteins. These patterns are
similar to those observed earlier in wild-type CheZ by Stock and Stock (30) . In contrast, CheZ110LP was significantly less resistant
to trypsin proteolysis and was almost completely degraded to small
fragments already within 1.5 min of incubation with trypsin; the
wild-type protein and the other mutant proteins remained almost intact
during this time period. This result suggests that the 110LP mutation
causes global perturbation in the structure of CheZ.
Figure 6:
Sensitivity of mutant CheZ proteins to
proteolysis by trypsin. CheZ was incubated with trypsin as described
under ``Experimental Procedures'' and analyzed at the
indicated time points.
DISCUSSION
In this study we demonstrated that the phosphatase activity
of CheZ is correlated with its ability to oligomerize upon interaction
with CheY
P. The study also confirmed the correlation between the
phosphatase activity of CheZ and the CCW bias of flagellar rotation,
and it provided an insight into the involvement of specific CheZ
residues in the functions of the protein. These issues are discussed
below.
Relation between CheZ-CheY
P Binding, Direction of
Flagellar Rotation, and Phosphatase Activity
We studied four
mutant CheZ proteins that in vivo are unable to antagonize the
CW causing activity of CheY
P(28) . We observed that all
these mutant proteins were, on the one hand, severely impaired in their
ability to dephosphorylate CheY (Fig. 3) but, on the other hand,
apparently normal in their ability to bind to CheY (Fig. 4).
This indicates that CheZ-CheY
P binding is not sufficient for
CheY
P dephosphorylation. The observation that CW biased cheZ mutants are defective in the phosphatase activity of CheZ (this
study) taken together with the observation that CCW biased cheZ mutants have phosphatase activity higher than wild-type CheZ (31) indicates that the direction of flagellar rotation is
tightly dependent on the phosphatase activity of CheZ.
Involvement of Specific CheZ Residues in the Functions of
the Protein
Three of the four mutant alleles of CheZ were
clustered between residues 141 and 145. These mutant proteins were
normal in binding CheY
P (Fig. 4) but were severely impaired
in their ability to oligomerize in its presence (Fig. 5).
Furthermore, even conservative substitutions such as Phe
Ile (at
position 141) or Asp
Glu (at position 143) were sufficient for
CheZ inactivation. This suggests that the region defined by residues
141-145 plays an important role in mediating CheZ oligomerization
and CheY
P dephosphorylation, but is not necessary for the binding
to CheY
P. Indeed, this region is part of a conserved domain in
CheZ of E. coli, S. typhimurium, and Pseudomonas
aeruginosa(30, 32, 33) . The
substitution Leu
Pro (at position 110) in the fourth mutant was
also a substitution of a conserved residue. This mutation had
apparently a global effect on the structure of CheZ, as evident from
the increased susceptibility of the protein to proteolysis (Fig. 6). Since proline residues are known to break
-helices, it is possible that Leu
is in an
-helix crucial for the structure of CheZ. In accordance with a
global structural effect, the mutation in residue 110, unlike the
mutations in residues 141-145, did not have an ``all or
none'' effect on the oligomeric state (Fig. 5). This may be
attributed to the indirect effect of this mutation on the domain
responsible for the oligomerization.
Correlation between the Phosphatase Activity and the
Oligomerization of CheZ
All the mutant CheZ proteins that we
studied were defective in both the phosphatase activity and the
oligomerization ( Fig. 4and Fig. 5), suggesting that
oligomerization is required for the expression of the phosphatase
activity. This suggests that the phosphatase activity of CheZ, like the
oligomerization of CheZ (27) , is dependent on the level of
CheY phosphorylation. This is in line with an earlier observation
(which was not understood at the time) that, when the level of CheY
phosphorylation is very low (
1%), it is essentially independent of
the CheZ concentration (in the range 5-100
µM)(26) . This earlier observation that CheZ is
not active at low levels of CheY phosphorylation, taken together with
the observations of this study, suggests that the phosphatase activity
is positively regulated by the level of CheY phosphorylation.
What Is the Physiological Role of CheZ
Oligomerization?
A reasonable possibility is that any modulation
of the level of CheY phosphorylation by chemotactic stimuli is
counterbalanced by a delayed modulation of the phosphatase activity of
CheZ. The delay may be caused by the oligomerization, which is
presumably rate-limiting. Thus, according to this possibility, an
increase in the level of CheY phosphorylation would increase the CW
bias of flagellar rotation and, in parallel, would cause a relatively
slow oligomerization of CheZ with a resultant increased phosphatase
activity and a decreasing CW bias (Fig. 7). Conversely, a
decrease in the level of CheY phosphorylation would decrease the CW
bias and, in parallel, would cause a relatively slow dissociation of
the CheZ oligomer with a resultant decreased phosphatase activity and
increased CW bias. This proposed adaptation mechanism would ensure that
the phosphorylation level is partially set back close to the
prestimulus level. Such a mechanism may be part of the
methylation-independent adaptation (34, 35, 36) (see for review, (3) and (37) ). The notion of CheZ involvement in
adaptation is well in line with earlier in vivo studies in
which cheZ mutants were found to adapt to both attractants (28, 35, 38) and repellents ( Fig. 7versus Fig. 10 in (39) ) significantly
slower than wild-type bacteria.(
)
Figure 7:
A simplified scheme of a proposed sequence
of events involving CheZ oligomerization. See text for details. Small up and down arrows represent an increase or a
decrease, respectively.
Note Added in
Proof-Recently Wang and Matsumura (Mol.
Microbiol.(1996) in press) found, in line with our observations
and suggestion that CheZ oligomerization increases its phosphatase
activity, that the multimeric complex between CheZ and the short form
of CheA has an increased phosphatase activity.