From the School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400 076, India
Received for publication, June 11, 2002, and in revised form, October 30, 2002
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ABSTRACT |
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The polymerization of FtsZ is a finely regulated
process that plays an essential role in the bacterial cell division
process. However, only a few modulators of FtsZ polymerization are
known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and
extent of polymerization of FtsZ in a
concentration-dependent manner; ~90% of the protein was
sedimented as polymer in the presence of 1 M
glutamate. Electron micrographs of glutamate-induced polymers showed
large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers;
interestingly, although 1 M glutamate produced a larger
light-scattering signal than produced by 10 mM calcium, the
amount of polymer sedimented in the presence of 1 M
glutamate and 10 mM calcium was similar. Thus, the
increased light scattering in the presence of glutamate must be due to
its ability to induce more extensive bundling of FtsZ polymers than
calcium. The data suggest that calcium and glutamate might induce FtsZ
polymerization by different mechanisms.
FtsZ, a 40.3-kDa protein, is a key protein involved in prokaryotic
cell division (1, 2). It forms the structural element called Z-ring at
the site of cell division, and it remains associated with the inner
face of the cytoplasmic membrane throughout the septation process (3).
Homologs of FtsZ have been found in every free-living prokaryote
examined to date (4-6), including many species of Archaea. It
has also been found in chloroplasts of higher plants, where it is
involved in chloroplast division (7). Its wide distribution and high
degree of sequence conservation suggest that it probably plays a
similar role in all bacterial and archaeal species (8-10).
Consistent with its cytoskeletal role, FtsZ has several
properties in common with the eukaryotic cytoskeleton protein, tubulin. Like tubulin, FtsZ binds and hydrolyzes GTP, and it polymerizes to form
long tubules in a GTP-dependent manner (11-14). It has been shown that purified FtsZ polymerizes into structures that closely
resemble those formed by purified tubulin (15). Furthermore, FtsZ shows
limited but significant sequence similarity to tubulin. Sequence
alignment of FtsZ and tubulin reveals that several structural glycine
and proline residues and most of the residues that are involved in GTP
binding are conserved. In addition, FtsZ contains the tubulin signature
GTP binding sequence motif GGGTG(T/S)G (11, 12, 16). Recently, the
crystal structure of FtsZ from Methanococcus jannaschii has
been determined (17). FtsZ has two domains arranged around a central
helix. GTPase activity is localized in the amino terminus domain of the
protein, whereas the carboxyl-terminal domain function is still unknown
(17). The three-dimensional structure of FtsZ also shows striking
similarities with At low concentrations, purified FtsZ monomers polymerize into
single-stranded protofilaments with little or no bundling of protofilaments in an assembly reaction that is believed to be isodesmic
(non-cooperative) in nature (19). However, in the presence of
DEAE-dextran (15, 20) or high concentrations of calcium (21-23) FtsZ
monomers polymerize into long, rod-shaped or tubular polymers that
become extensively bundled. The assembly reaction is stimulated, and
resulting polymers are stabilized by these agents. These studies
demonstrate that the extent and nature of the polymer formed highly
depends on the reaction conditions. Interestingly, calcium exerts
contrasting effects on the polymerization of FtsZ and microtubules.
Calcium strongly inhibits tubulin assembly and depolymerizes
microtubules, whereas it promotes FtsZ assembly and induces bundling of
FtsZ filaments (23). Biochemical studies have been carried out to study
the effect of various factors such as temperature and pH on FtsZ
polymerization (24-26). Many inducers of tubulin polymerization have
been identified over the years, and these modulators of polymerization
have been extensively utilized to understand the molecular mechanism of
microtubule polymerization dynamics (27); however, only a few inducers
of FtsZ polymerization have thus far been identified.
In this study we found that monosodium glutamate is a potent inducer of
FtsZ polymerization, and it stabilizes FtsZ polymers against
disassembly. Electron micrographs showed that glutamate-induced FtsZ
polymers form extensive bundles. Furthermore, glutamate decreased the
GTPase activity of FtsZ. The property of filament bundling may be
important in FtsZ function in the septation process. In addition,
calcium and glutamate exerted differential effects on FtsZ
polymerization, indicating that they modulate FtsZ polymerization through different mechanisms.
Materials--
Pipes,1
monosodium glutamate,
isopropyl- Purification of FtsZ--
Recombinant Escherichia
coli FtsZ was overexpressed and purified from BL21 strain (a gift
from Dr. H. P. Erickson, Duke University) as described earlier
(28) with some modifications. Briefly, the cells were grown at 37 °C
in LB broth containing 50 µg/ml ampicillin until they reached early
log phase (A600 ~ 0.4-0.6) when protein
expression was induced by the addition of 0.4 mM isopropyl- Light-scattering Assay--
FtsZ (6 µM) was
prepared in 25 mM Pipes buffer (pH 6.8) and 3 mM MgSO4 (buffer A) in the absence and presence
of different concentrations of monosodium glutamate on ice. After the
addition of 1 mM GTP, the sample was immediately placed in
a cuvette at 37 °C, and the polymerization reaction was monitored at
37 °C by light scattering at 500 nm using a JASCO 6500 spectrofluorometer. The excitation wavelength was 500 nm.
Sedimentation Assay--
FtsZ (6 µM) was
polymerized in buffer A containing 1 mM GTP in the absence
and presence of different concentrations of sodium glutamate for 30 min
at 37 °C. The polymers were collected by sedimenting them at
27,000 × g for 30 min at 27 °C. The protein concentration in the supernatant was measured by the Bradford assay
using bovine serum albumin as a standard.
Electron Microscopy--
FtsZ (6 µM) was
polymerized at 37 °C in buffer A with 1 mM GTP and in
the absence or presence of 1 M glutamate for 30 min. The
FtsZ polymers in the samples were fixed with prewarmed 0.5% glutaraldehyde for 5 min. Twenty-microliter volumes of protein samples
were transferred onto carbon-coated copper grids (300 mesh) and blotted
dry. The grids were subsequently negatively stained with a few drops of
2% uranyl acetate solution and air-dried. The samples were examined
using a JEOL-JEM 100S or a Philips CM 200 electron microscope.
Stability of FtsZ Polymers in the Presence of
Glutamate--
FtsZ (1 mg/ml) was polymerized in buffer A with 10 mM calcium and 1 mM GTP at 37 °C for 30 min.
The protein solution was then diluted 20 times to reach a final FtsZ
concentration of 0.05 mg/ml in different concentrations of warm
glutamate. The reaction mixtures were incubated at 37 °C for another
30 min and centrifuged at 50,000 × g for 30 min. The
pellet obtained were dissolved in 30 µl of 25 mM Pipes
buffer containing 0.5% SDS, and 20 µl of each sample was analyzed by
10% SDS-PAGE. The intensity of the bands was determined by a gel
documentation system obtained from Kodak Digital Science.
GTP Hydrolysis--
A malachite green sodium molybdate assay was
used to measure the production of inorganic phosphate during GTP
hydrolysis (19, 30). Samples containing 6 µM FtsZ with
different concentrations of glutamate were prepared in buffer A at
0 °C, and 1 mM GTP was added to the reaction mixtures.
Immediately, the reaction mixtures were transferred to 37 °C and
incubated for different lengths of time. The hydrolysis reaction was
quenched at desired time points by the addition of 10% v/v 7 M perchloric acid to the reaction mixtures, and the
quenched reaction mixtures were centrifuged for 5 min to remove
aggregated proteins. Twenty microliters of the supernatants were
incubated with 900 µl of filtered malachite green solution (0.045%
malachite green, 4.2% ammonium molybdate, and 0.02% Triton X-100) at
room temperature for 30 min, and the phosphate ions produced were
determined by measuring the absorbance of samples at 650 nm. The
reaction was normalized including a control without FtsZ. A phosphate
standard curve was prepared using sodium phosphate.
Purification of FtsZ by Glutamate-enhanced
Polymerization--
FtsZ and its eukaryotic homolog tubulin are both
known to undergo multiple temperature-dependent
polymerization and depolymerization cycles in the presence of GTP (31).
The polymerization of tubulin is well understood, and several inducers
of tubulin assembly in vitro are known. The purification
procedure for tubulin involves temperature- and
GTP-dependent polymerization and depolymerization cycles
followed by phosphocellulose chromatography, which not only increases
the purity level of the protein but also increases the yield of the
assembly-competent fraction of tubulin (32, 33). Taking advantage of
the tubulin literature, we sought to find inducers of FtsZ
polymerization. We tried several well known agents that promote tubulin
polymerization including dimethyl sulfoxide (Me2SO),
glycerol, taxol, and glutamate (34-36). In contrast to their effects
on tubulin polymerization, 4 M glycerol, 10% v/v
Me2SO, and 10 µM taxol did not show any
effect on FtsZ polymerization (data not shown). However, glutamate did
enhance FtsZ assembly in a concentration range similar to the
concentration range that enhances tubulin polymerization
(Fig. 1). Several groups have routinely purified FtsZ from crude extracts using a cation exchange (DE-52) chromatographic procedure (13, 28). Thus, similar to the
purification procedure used for tubulin, we introduced a
glutamate-induced temperature-dependent polymerization and
depolymerization cycle after the DE-52 column to further purify FtsZ
while keeping its activity intact ("Experimental Procedures"). The
combination of a cycle of polymerization and depolymerization with
DE-52 chromatography yielded FtsZ of high purity (>98%) as analyzed
by Coomassie-stained SDS-PAGE (data not shown).
Glutamate-induced Polymerization of FtsZ--
Our goal was to
discover modulators of FtsZ polymerization, which could be further used
to understand the basic mechanisms of FtsZ polymerization and the roles
of FtsZ polymerization in bacterial cell division. Monosodium glutamate
increased the rate and extent of FtsZ polymerization in a
concentration-dependant manner with an optimal effect at a
concentration of 1 M (Fig. 1A). To further
demonstrate that glutamate is a potent inducer of FtsZ polymerization,
the rate and extent of FtsZ (8 µM) polymerization was
first monitored for 10 min by light scattering in the absence of
glutamate. After 10 min, 1 M glutamate was added to the
cuvette from a stock of 2 M warm glutamate, and the
polymerization reaction was monitored for an additional 15 min. The
addition of 1 M warm glutamate strikingly increased the
magnitude of the light-scattering signal, and an apparent steady state
was achieved quickly (Fig. 1B).
To verify whether the increase in the light-scattering signal was due
to a genuine increase in the FtsZ polymerization, we measured the mass
of polymeric FtsZ in the presence of different concentrations of
glutamate (Fig. 2). The FtsZ polymer mass
clearly increased with increasing concentrations of glutamate. Between 85 and 92% of the initial protein could be sedimented as polymer mass
in the presence of 1 M glutamate. To find out whether
glutamate-induced FtsZ polymerization was
temperature-dependent or not, 6 µM FtsZ was
incubated with 1 M glutamate plus 1 mM GTP in
buffer A for 30 min at 4 °C. After incubation, the reaction mixture
was centrifuged at 4 °C as previously described in Fig. 2. Then the
protein concentration of the supernatant was measured, and it was found
to be identical with the total protein concentration used in the assay
(data not shown). The result showed that no glutamate-induced
polymerization occurred at 4 °C. However, ~90% of the total
protein formed sedimentable polymers at 37 °C, indicating that the
polymerization reaction is temperature-dependent.
Electron micrographic analysis of the FtsZ polymer showed formation of
a dense network of FtsZ polymers in the presence of 1 M
glutamate (Fig. 3, B and
C), whereas only a few polymeric filamentous structures were
formed in the absence of glutamate (Fig. 3, A and
D). Furthermore, analysis of the electron micrographs showed
that sodium glutamate not only increased the amount of polymer but also
induced formation of large filamentous polymeric structures with
extensive bundle formations (Fig. 3, E and
F).
Does Glutamate Stabilize FtsZ Polymers against Dilution-induced
Disassembly?--
We wanted to determine whether glutamate could
stabilize FtsZ polymers against dilution-induced disassembly. FtsZ (1 mg/ml) was initially polymerized with 10 mM calcium in the
presence of 1 mM GTP. The polymerized FtsZ was diluted 20 times to a final concentration of 0.05 mg/ml FtsZ into buffers
containing different concentrations of warm monosodium glutamate at
37 °C. After 30 min of incubation, FtsZ polymers were sedimented,
and the protein concentrations in the pellets were quantified using
Coomassie-stained SDS-PAGE (Fig. 4,
A and B), which clearly showed that the amount of
polymeric protein increased with increasing glutamate concentration. For example, the polymer content increased by 9.2-fold in the presence
of 1 M glutamate as compared with the control value. Thus,
the data indicated that glutamate stabilizes FtsZ polymers and prevents
their dilution-induced disassembly.
Glutamate Reduces the GTPase Activity of FtsZ--
FtsZ is a
GTPase, and the polymerization of FtsZ is thought to be regulated by
GTP hydrolysis (14, 37). Initially, we wanted to know whether GTP is
essential for glutamate-induced FtsZ polymerization. The FtsZ
polymerization reaction was carried out for 10 min in the presence of 1 M glutamate without GTP. There was no development of light
scattering (Fig. 5A). After 10 min, 1 mM GTP was added to the cuvette, and the
polymerization was immediately started, demonstrating that GTP is
required for glutamate-induced polymerization of FtsZ. Similarly, GTP
was also found to be essential for calcium-induced polymerization of
FtsZ (Fig. 5A). Because GTP was found to be essential for
the glutamate-induced assembly of FtsZ, we sought to determine the
effects of glutamate on the GTPase activity of FtsZ. Monosodium
glutamate decreased the rate of GTP hydrolysis in a
concentration-dependent manner (Fig. 5B). For
example, 1 M glutamate reduced the amount of hydrolyzed GTP of FtsZ by ~50%. Interestingly, GTP hydrolysis occurred at a
significantly higher rate in the initial stage of the reaction than the
late stage of the reaction (Table I). For
example, in the absence of glutamate, 5 and 30 min of the hydrolysis
reaction produced 69 ± 11 and 103 ± 8 mol of phosphate
ions, respectively. However, in the presence of 1 M
glutamate, 36 ± 7 and 56 ± 4 mol of phosphate ions were
produced after 5 and 30 min of hydrolysis reaction (Table I). Glutamate
stabilized the FtsZ polymers (Fig. 4, A and B) and induced an extensive bundling of FtsZ polymers (Fig. 3,
E and F); thus, the reduction in the GTPase
activity of FtsZ polymers by monosodium glutamate could be due to the
enhanced stability of the FtsZ polymers.
Comparison of the Effects of Glutamate and Calcium on FtsZ
Polymerization--
Recently, it has been shown that high millimolar
concentrations of calcium enhance the rate and the extent of FtsZ
polymerization and promote the formation of tubulin-like protofilaments
in the polymers (21, 22). Thus, it was interesting to compare the effects of glutamate and calcium on FtsZ polymerization. As previously reported, we also found that calcium induced polymerization of FtsZ, as
evident from the increase in light scattering (Fig.
6A). However, glutamate (1 M) strikingly increased the magnitude of light scattering
compared with 10 mM calcium (Fig. 6A). The
increased light scattering by glutamate compared with calcium might be
due to an increase in the polymer mass, to changes in the size and shape of the polymers, or to a combination of both the factors. To
discern the possibilities, a sedimentation assay and electron microscopy were performed. We found that similar amounts of polymeric FtsZ were sedimented when the polymerization reaction was initiated by
either 10 mM calcium or 1 M glutamate (Fig.
6B). Thus, the observed increase in the light-scattering
intensity in the presence of glutamate was not due to an increase in
the polymer mass. The electron micrographs of FtsZ polymers formed in
the presence of 10 mM calcium are shown in Fig.
6C. These polymers are similar to previously reported
calcium-induced FtsZ polymers (21). However, large filamentous
structures containing thick bundles of FtsZ polymers were more abundant
in the presence of 1 M glutamate (Fig. 3) compared with
calcium (Fig. 6C). Therefore, the observed increase in light
scattering in the presence of glutamate must be due to extensive
bundling of FtsZ polymers.
In this report we found that monosodium glutamate increased the
rate and extent of FtsZ polymerization. It also enhanced the bundling
of FtsZ filaments, and it stabilized the polymers against dilution-induced disassembly. At low protein concentrations, FtsZ assembly reaction is believed to be isodesmic (non-cooperative). Under
these conditions, FtsZ monomers assemble into single-stranded polymers
with few protofilament bundles (19). However, polycations such as
DEAE-dextran and calcium have been shown to modify the morphology of
FtsZ polymers. In the presence of DEAE-dextran, FtsZ protofilaments
associate to form three-dimensional sheets (20, 38) and millimolar
concentrations of calcium-pronounced bundling of FtsZ filaments have
been observed by several groups (21-23). It was suggested that
DEAE-dextran and calcium increase the lateral interactions between the
protofilaments (20, 39).
Hydrophobic interactions play an important role in FtsZ assembly. It
was proposed previously that drugs like
bis-1-anilino-8-naphthalenesulfonate inhibits FtsZ polymerization,
presumably by blocking intermolecular hydrophobic interactions between
FtsZ molecules (40), whereas binding of calcium or DEAE dextran to FtsZ
increases the intermolecular hydrophobic interactions, resulting in
stimulation of FtsZ assembly (22, 38). In our experiments we found that
the glutamate increased the rate and extent of the FtsZ polymerization
to an optimal effect at a concentration of 1 M. At the
optimal concentration of glutamate, FtsZ polymerization was biphasic,
with a rapid burst phase of polymerization followed by a slower and
prolonged linear phase (Fig. 1A). Electron micrographs of
glutamate-enhanced FtsZ polymers showed large filamentous structures
with extensive bundles (Fig. 3). These polymeric structures were
somewhat similar to the FtsZ polymers formed in the presence of high
concentrations of calcium (22); however, more bundles were present in
the presence of glutamate compared with calcium. The results indicated
that, like calcium, glutamate could increase the intermolecular
hydrophobic interactions in FtsZ and stabilize the polymers by
enhancing bundle formation. The slower phase of polymerization observed
by light scattering could be due to the formation of the FtsZ polymer
bundles. Furthermore, when preformed FtsZ polymer was diluted into
different concentrations of glutamate, the polymers were found to be 9 times more stable in the presence of 1 M glutamate compared
with no glutamate (Fig. 4). This shows that glutamate not only enhanced the FtsZ polymer formation but also stabilizes the preformed polymers.
Kinetics of nucleotide hydrolysis is important to describe the model
for FtsZ polymerization. The active site for GTP hydrolysis in FtsZ is
postulated to form when the T7 loop of a FtsZ monomer binds with the
nucleotide-binding site on an adjacent FtsZ monomer (41). It was also
thought that, like microtubules, FtsZ polymers could also be stabilized
by a GTP cap (39). But recently Scheffers and Driessen (42) showed that
GTP hydrolysis occurs immediately after the addition of GTP to FtsZ,
and FtsZ polymers contain mostly GDP and inorganic phosphate rather
than GTP (42). FtsZ assembly does not require magnesium or calcium, but
magnesium is required for dynamic behavior of FtsZ polymers (43),
whereas calcium at high concentrations induces the bundling of
polymers, which reduces the dynamic behavior of FtsZ assembly (23). In
the absence of magnesium, calcium inhibits GTP hydrolysis and reduces
the dynamic nature of FtsZ polymers by making the polymers more stable. Our experiment showed that glutamate decreased the GTPase activity of
FtsZ (Fig. 5). Glutamate might stabilize FtsZ polymers by increasing hydrophobic interactions between subunits and enhancing bundle formation, which decreased the dynamics of the polymers. Thus, the
increased stability of FtsZ could reduce the rate of subunit exchange,
which might be the cause of suppression of its GTPase activity.
However, it is also difficult to rule out the possibility that the
reduction in the GTPase activity of FtsZ by glutamate could also be
responsible for higher stability of the polymers.
Calcium induces polymerization of FtsZ (Fig. 6 and Ref. 21).
Interestingly, glutamate strikingly increased the rate and extent of
the development of the light-scattering signal of FtsZ polymerization
compared with calcium (Fig. 6A). However, the polymer mass
was found to be similar in the presence of either glutamate or calcium.
Electron micrographs of FtsZ polymers showed that glutamate caused
extensive bundling of polymers compared with the degree of bundling
caused by calcium, indicating that glutamate induces different
conformational changes in the polymer than does calcium. Because 10 mM calcium alone was sufficient to polymerize most of the
FtsZ, the increased intensity in the light scattering signal during
FtsZ polymerization in the presence of 1 M glutamate must
be due to the rearrangement of FtsZ polymers into thicker bundles
rather than to an increase in polymer mass. The data indicate not only
the presence of different modulator sites on FtsZ but also different
mechanisms of FtsZ polymerization.
Tubulin, the eukaryotic homolog of FtsZ, is also known to
form different types of polymers, including microtubules, open sheets (in the presence of zinc and Me2SO), double rings and
isodesmic aggregates (in the presence of vinblastine), although only
microtubules are responsible for the functional properties of tubulin
(34, 44, 45). Interestingly, we found that the common inducers of
tubulin assembly such as Me2SO, glycerol, and taxol had no effect on FtsZ polymerization (data not shown). It has been reported that high concentrations of glutamate stabilize tubulin and induce its
polymerization. Glutamate increases the GTPase activity in tubulin and
thereby enhances the rate of polymerization of tubulin into
microtubules (36). In contrast, we found that glutamate strongly
suppressed the GTPase activity of FtsZ. The binding of glutamate to
tubulin is thought to induce localized opening or disordering of
tubulin subunits without any major reorganization of the internal
structure of tubulin (46). These data together indicate that glutamate
interacts with FtsZ in a manner different from that of tubulin or microtubules.
FtsZ polymerizes to form a dynamic ring marking the division plane of
prokaryotic cells and plays an essential role in cytokinesis (1, 3).
Despite its actin-like role, FtsZ has structural and biochemical
similarity to tubulin (5, 11, 15, 17). Tubulin, the eukaryotic homolog
of FtsZ, has been successfully targeted for the development of
anticancer and antifungal drugs (47-50). Thus, the potential use of
FtsZ as a drug target for the development of antimicrobial agents needs
to be explored. Several potent inhibitors of tubulin assembly including
colchicine and vinblastine have no effect on FtsZ assembly, suggesting
that these drugs do not interact with FtsZ (40). Interestingly, calcium depolymerizes microtubules (51), whereas it promotes the polymerization of FtsZ. Furthermore, glycerol, Me2SO and taxol are well
characterized inducers of microtubule assembly, but these agents do not
induce FtsZ assembly, suggesting that the polymerization reaction of FtsZ is mechanistically different from that of tubulin assembly. The
finding of new modulators of FtsZ assembly-like glutamate would
certainly help to characterize FtsZ polymerization and may help to
search for new inhibitors. For example, a screen could be designed in
which inhibitors of glutamate-induced polymerization might be identified.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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and
tubulin (18). Furthermore, molecular
phylogenetic data indicate that an archaeal FtsZ is the most
tubulin-like of all prokaryotic FtsZ proteins found so far, suggesting
that tubulin may have evolved from an ancestral FtsZ (5).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES
-D-thiogalactopyranoside, bovine serum
albumin, and GTP were obtained from Sigma. DE-52 was from Whatman
International Ltd. All other chemicals used were of analytical grade.
-D-thiogalactopyranoside for an additional
5 h. Cells were harvested by centrifugation at 12,000 × g at 4 °C for 30 min and suspended in lysis buffer (50 mM Tris (pH 8), 100 mM NaCl, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1%
-mercaptoethanol, and 1 mM MgSO4) on ice.
Cells were lysed by sonication with 20-s pulses at 30-s intervals
between the pulses for four cycles. Partially lysed cells were further
disrupted using a Polytron homogenizer (Polytron PT-3000). Lysozyme
(0.4 mg/ml) was added to the partially lysed cell suspension, which
then was incubated on ice for 1 h. It was again incubated with 10 mM MgSO4 for 5 min, and the suspension was
sonicated for 6 cycles with 20-s pulses. The insoluble debris was
removed by centrifugation at 12,000 × g at 4 °C for
30 min. The cell-free supernatant was saturated to 25% with ammonium
sulfate solution and incubated for 90 min at 4 °C. The protein
suspension was centrifuged at 27,000 × g at 4 °C
for 30 min. The pellet was resuspended in 50 mM Tris buffer
(pH 8) and dialyzed against the same buffer for 4 h. Purification
of FtsZ was further carried out using DE-52 ion exchange column
chromatography as described previously (13). Briefly, FtsZ was loaded
on a DE-52 column pre-equilibrated with 50 mM Tris buffer
(pH 8), and the protein was eluted with increasing concentrations of
KCl. FtsZ-containing fractions were eluted at 250 mM KCl;
protein concentrations were determined by the Bradford method (29).
These fractions were pooled together and concentrated using Amicon
Centriplus concentrators at 4 °C. One cycle of
temperature-dependent polymerization and depolymerization
was then used to further purify the FtsZ. The polymerization reaction
was carried out in buffer containing 25 mM Pipes (pH 6.8),
10 mM MgSO4, 10 mM
CaCl2, 1 M monosodium glutamate, and 1 mM GTP at 37 °C for 30 min. FtsZ polymers were
sedimented by centrifugation at 27,000 × g for 30 min
at 27 °C. The pellet was resuspended on ice in 25 mM
Pipes buffer (pH 6.8), and the suspension was centrifuged at
12,000 × g for 15 min at 4 °C to remove the
insoluble aggregates. The FtsZ concentration was determined by the
Bradford method using bovine serum albumin as a standard (29), and FtsZ
was frozen and stored at
80 °C.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 1.
Glutamate-induced polymerization of
FtsZ. A, FtsZ (6 µM) was polymerized in
buffer A containing 1 mM GTP at 37 °C, in the absence
( ) and presence of 0.25 (
), 0.5 (
), 0.75 (
), and 1 M (
) glutamate. The rate and extent of the
polymerization reaction were measured by light scattering at 500 nm
using a fluorescence spectrophotometer. The light-scattering signals
were corrected by subtracting appropriate blank scans. B,
FtsZ (8 µM) was polymerized in buffer A containing 1 mM GTP at 37 °C for 10 min. Then 1 M
glutamate was added to the cuvette, and the polymerization of FtsZ was
monitored for an additional 15 min. The glutamate solution was added
from a stock solution of 2 M glutamate, which reduced the
concentration of FtsZ to 4 µM. The light-scattering data
were corrected with respect to buffer A and 1 M
glutamate.
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Fig. 2.
Determination of polymer mass by a
sedimentation assay. FtsZ (6 µM) was polymerized in
buffer A containing 1 mM GTP in the absence and presence of
different concentrations of glutamate at 37 °C for 30 min and
sedimented at 27,000 × g at 25 °C for 45 min. The
protein concentrations of the supernatants were determined by the
Bradford method. The protein concentrations of the pelleted fractions
were determined by subtracting the supernatant concentrations from the
total protein concentrations. Data were averages of three
independent experiments.
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Fig. 3.
Electron micrographs of FtsZ polymers.
FtsZ (6 µM) was polymerized in buffer A in the absence or
presence of 1 M glutamate at 37 °C. The samples were
prepared for electron microscopy as described under "Experimental
Procedures." A (×10,000) and D (×28,000) show
FtsZ polymers in the absence of glutamate and B and
C (×10,000) and E and F (×28,000)
show FtsZ polymers formed in the presence of 1 M
glutamate.
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Fig. 4.
Stabilization of FtsZ polymers against
dilution-induced disassembly by glutamate. FtsZ (1 mg/ml) was
polymerized in the presence of 10 mM calcium and 1 mM GTP for 30 min. Polymerized FtsZ was diluted 20 times
into buffer A containing different concentrations (0, 0.25, 0.5, 0.75, and 1 M) of glutamate at 37 °C. Samples were
centrifuged, dissolved, and loaded on a 10% SDS-PAGE as described
under "Experimental Procedures." A, the intensities of
the bands were determined by a gel documentation system from Kodak
Digital Science, and relative protein concentrations of these bands
were determined with respect to control. The data were plotted against
glutamate concentrations (B).
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Fig. 5.
A, FtsZ (6 µM) was
polymerized in buffer A in the presence of either 1 M
glutamate ( ) or 10 mM CaCl2 (
) without
GTP, and the assembly reaction was monitored for 10 min by light
scattering 500 nm. After 10 min of reaction as marked by an
arrow, 1 mM GTP was added to the cuvette, and
the polymerization reaction was followed for an additional 10 min.
B, effects of glutamate on the GTPase activity of FtsZ. FtsZ
(6 µM) was mixed with different concentrations of
glutamate on ice. After the addition of 1 mM GTP to the
reaction mixtures, the sample solutions were immediately transferred to
37 °C and incubated for 5 min. The extent of GTP hydrolysis was
measured as described under "Experimental Procedures." The data are
averages of three experiments.
Effects of glutamate on the GTPase activity of FtsZ
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Fig. 6.
A, FtsZ (6 µM)
was polymerized in the presence of either 10 mM
CaCl2 ( ) or 1 M glutamate (
).
The control polymerization reaction (
) was carried out in buffer A
in the absence of both CaCl2 and glutamate. The
polymerization reaction was monitored by light scattering at 500 nm.
B, after 30 min of polymerization at 37 °C, FtsZ polymers
were centrifuged at 50, 000 × g, and pellets were
dissolved in 25 mM Pipes buffer containing 0.5% SDS. The
protein content of the pellets were determined by Coomassie staining of
a 10% SDS-PAGE. Lanes 1 and 2 show the
Coomassie-stained gel photographs of FtsZ sedimented in the presence of
10 mM calcium or 1 M glutamate, respectively.
C, electron micrographs of FtsZ polymers. FtsZ (6 µM) was polymerized in the buffer A in the presence of 10 mM calcium at 37 °C. The samples for electron microscopy
were prepared as described under "Experimental Procedures," and the
pictures were taken at ×27,500 magnifications.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. H. P. Erickson for providing us the FtsZ clone. We thank Drs. Leslie Wilson, D. Dasgupta, and S. Pathare for critical reading of the manuscript. We thank the Regional Sophisticated Instrumentation Centre, IIT, Bombay and the Tata Institute of Fundamental Research, Bombay for allowing us to use their electron microscopy facilities.
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FOOTNOTES |
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* The study was funded by a grant (to D. P.) from the Department of Science and Technology, Government of India.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.
Supported by fellowship from Council of Scientific and Industrial
Research, Government of India.
§ To whom correspondence should be addressed. Tel.: 91-22-572-2545 (ext. 7838); Fax: 91-22-572-3480; E-mail: panda@btc.iitb.ac.in.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M205760200
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ABBREVIATIONS |
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The abbreviation used is: Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid).
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REFERENCES |
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