Molecular Microbiology (2001) 39(4), 949959

Cell cycle and positional constraints on FtsZ

localization and the initiation of cell division in
Caulobacter crescentus
Ellen M. Quardokus,1 Neena Din2 and Yves V. Brun1*
1
Department of Biology, Jordan Hall 142, Indiana
University, 1001 E. 3rd St., Bloomington, IN 47405-3700,
USA.
2
Department of Biology, Loyola College, Baltimore, MD
21210, USA.
Summary
Swarmer cells of Caulobacter crescentus are devoid of
the cell division initiation protein FtsZ and do not
replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked
cells. We show that FtsZ first localizes at the incipient
stalked pole in differentiating swarmer cells. FtsZ
subsequently localizes at the mid-cell early in the cell
cycle. In an effort to understand whether Z-ring
formation and cell constriction are driven solely by
the cell cycle-regulated increase in FtsZ concentration,
FtsZ was artificially expressed in swarmer cells at a
level equivalent to that found in predivisional cells.
Immunofluorescence microscopy showed that, in
these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring
formation took place only in stalked cells. Expression
of FtsZ in swarmer cells did not alter the timing of cell
constriction initiation during the cell cycle but,
instead, caused additional constrictions and a delay
in cell separation. These additional constrictions were
confined to sites close to the original mid-cell
constriction. These results suggest that the timing
and placement of Z-rings is tightly coupled to an early
cell cycle event and that cell constriction is not solely
dependent on a threshold level of FtsZ.

Introduction
The earliest known step defined for the initiation of bacterial
cell division is the polymerization of the GTPase FtsZ to
form a ring structure referred to as the Z ring at the future
site of cell division (Bi and Lutkenhaus, 1991). In
Escherichia coli, the Z-ring appears at approximately the
Accepted 15 November, 2000. *For correspondence. E-mail ybrun@
bio.indiana.edu; Tel. (11) 812 855 8860; Fax (11) 812 855 6705.
Q 2001 Blackwell Science Ltd

same time as the termination of DNA replication, but before

cell constriction and visible nucleoid separation can be
detected (Den Blaauwen et al., 1999). The Z-ring is
associated with the cytoplasmic membrane and constricts
as cell division proceeds (Lutkenhaus and Addinall, 1997;
Rothfield et al., 1999). In E. coli, FtsZ is required for the
localization of the other known cell division proteins to the
site of cell division (Rothfield et al., 1999) and, in part,
regulates the frequency of cell division (Bi and Lutkenhaus,
1990; Garrido et al., 1993; Palacios et al., 1996).
In the differentiating bacterium Caulobacter crescentus,
cell division is tightly coupled to the developmental
programme (Ohta and Newton, 1996; Brun and Janakiraman, 2000; Ohta et al., 2000). Each cell division
generates two progeny cells that differ in their morphology
and their ability to initiate DNA replication and cell division.
The non-motile stalked cell starts a new cell cycle
immediately after the previous cell division. In contrast,
the motile swarmer cell must undergo an obligatory gap
period, during which it is unable to begin a new cell cycle.
FtsZ levels are regulated in a cell cycle-dependent
manner in Caulobacter (Quardokus et al., 1996). Swarmer
cells are devoid of FtsZ. ftsZ transcription begins during
the differentiation of swarmer cells into stalked cells and,
at the same time, DNA replication is initiated. FtsZ is
stable during the assembly of the Z-ring and accumulates
until it reaches its maximal concentration at the initiation
of cell constriction, after which it becomes highly labile
(Quardokus et al., 1996; Kelly et al., 1998).
The timed expression of Caulobacter cell division genes
suggests that the developmental pathway may impose
restrictions on the time at which cell division can be
initiated (Sackett et al., 1998). The simplest model is that
a threshold concentration of FtsZ is both necessary and
sufficient to drive the initiation of Z-ring formation and cell
constriction. In this paper, we use synchronized swarmer
cells with a xylose-inducible ftsZ gene to investigate
whether this concentration model is correct. When ftsZ is
placed under the control of a xylose-inducible promoter in
the high-copy-number plasmid pUJ142 (Meisenzahl et al.,
1997), the concentration of FtsZ can be increased to
levels at least 10-fold higher than in wild-type cells (Din
et al., 1998). This level of FtsZ produces multiple cell
constrictions and transiently inhibits cell separation (Din
et al., 1998). In addition, high-level transcription of ftsZ in

950 E. M. Quardokus, N. Din and Y. V. Brun

swarmer cells can overcome the proteolysis of FtsZ and
leads to a substantial accumulation of FtsZ in these cells
(Kelly et al., 1998). We show that Z-rings are unable to
form in swarmer cells even when FtsZ is present at a
sufficient concentration at the beginning of the cell cycle.
Although Z-ring formation occurs slightly earlier in these
cells, the timing of cell constriction remains unaffected.
This indicates that other cell cycle events in addition to
FtsZ concentration are involved in controlling the timing of
cell constriction. We also show that, when FtsZ is
overproduced, the formation of additional Z-rings and
constrictions is confined to locations that are proximal to
the first Z-ring and never occurs at the poles of the cell.
These results indicate that Caulobacter cells exert a tight
temporal and positional control on these events.
Results
Quantification of FtsZ concentration during the cell cycle
As a baseline for FtsZ overexpression experiments, we
estimated the number of FtsZ molecules present in wildtype Caulobacter at different stages of the cell cycle. We
removed aliquots of cells from a synchronized population
of wild-type strain NA1000 at 15 min intervals and
compared the intensity of the FtsZ bands by immunoblot
with those of known amounts of purified FtsZ-His run on
the same gel. As shown previously (Quardokus et al.,
1996), the concentration of FtsZ was undetectable by
immunoblotting in newly harvested swarmer cells. At
30 min, before the differentiation of swarmer cells, there
were < 1000 molecules of FtsZ cell21 (Fig. 1B). After
60 min, soon after swarmer cell differentiation, the
concentration of FtsZ was < 6400 molecules cell21. The
maximum number of FtsZ molecules present in predivisional cells (90 min) is < 7600 molecules cell21. The
concentration of FtsZ subsequently decreased to < 1700
molecules cell21 at the time of cell division. Previous
studies have reported the concentration of FtsZ to be
between 15 000 and 20 000 molecules cell21 in log
phase E. coli cells (Dai and Lutkenhaus, 1992; Lu et al.,
1998). The lower concentration of FtsZ in Caulobacter
may reflect the smaller diameter of the cells.
Overproduction of FtsZ in swarmer cells and throughout
the cell cycle
In order to obtain swarmer cells containing a high
concentration of FtsZ, a culture of NA1000 containing
pUJ142ftsZ was grown exponentially for 2 h in both
induced (M2-GX) and uninduced (M2-G) conditions. The
concentration of FtsZ in uninduced cultures was the same
as in NA1000 without plasmid (Fig. 1A). Swarmer cells
were isolated from each culture, resuspended in M2-GX

Fig. 1. Variation in FtsZ during the Caulobacter cell cycle.

A. FtsZ concentration in NA1000 (wt), NA1000/pUJ142ftsZ grown
without xylose (un), NA1000/pUJ142ftsZ grown for 2 h with 0.3%
(w/v) xylose (in) in either M2G minimal medium or PYE complex
medium. Equal amounts of cells were loaded.
B. Cell cycle variation in FtsZ concentration. The graph shows the
variation in the number of FtsZ molecules in NA1000 (D), in an
uninduced culture of NA1000/pUJ142ftsZ (A) and in an induced
culture of NA1000/pUJ142ftsZ (X) as determined by densitometry
of immunoblots. The immunoblot below the graph shows the level
of FtsZ in uninduced and induced cultures. Aliquots of cells were
removed for immunoblotting analysis and protein determination at
the indicated times after the start of the experiment. Equal amounts
of protein were loaded in each lane. The 1 sign indicates samples
taken from cultures in which FtsZ expression was induced with
xylose, and indicates samples taken from uninduced cultures.
The maximum concentration of FtsZ in NA1000 and in NA1000/
pUJ142ftsZ was normalized to 100.

and M2-G, respectively, and allowed to proceed through

the cell cycle. Samples were removed every 15 min for
immunoblot analysis and microscopy. In the uninduced
culture, FtsZ levels throughout the cell cycle mimicked
FtsZ levels in NA1000 cells without pUJ142ftsZ (Fig. 1B).
No FtsZ was detectable in swarmer cells by immunoblot
(Fig. 1B). FtsZ was detected at 30 min and continued
to increase until 90 min into the cell cycle when it
reached its maximum level. In the induced cell population,
swarmer cells contained an FtsZ concentration equivalent
to the maximum concentration of FtsZ found in the
uninduced cells (Fig. 1B, compare 0 min1xylose with
90 minxylose). As induced cells progressed through the
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

Regulation of FtsZ ring formation in Caulobacter

cell cycle, the concentration of FtsZ continued to increase.
At 120 min, the concentration of FtsZ in the induced
population was < 3.5 times higher than in the uninduced
population. Thus, the concentration of FtsZ in induced
cells exceeded the concentration of FtsZ in uninduced
cells throughout the cell cycle. In addition, swarmer cells
from the induced culture contained a sufficient concentration of FtsZ to form a functional Z-ring.
FtsZ localizes to the incipient stalked pole early in the cell
cycle
FtsZ mid-cell localization can be detected in stalked cells
before cell constriction (Kelly et al., 1998). To determine
whether the timing of Z-ring formation depends on FtsZ
concentration, we used an affinity-purified anti-FtsZ antibody to localize FtsZ in cells overexpressing FtsZ. This
antibody cross-reacts specifically with FtsZ on immunoblots (Din et al., 1998). Control immunofluorescence
experiments using only the secondary antibody showed
only a very faint background fluorescence that was evenly
distributed in the cytoplasm (not shown). Cells were
collected every 15 min in each culture and stained to
follow the distribution of FtsZ. Early in the cell cycle, many
uninduced cells had a clear localization signal at a single
pole (Figs 2A and B and 3, second cell picture). This polar

951

localization signal appeared as a single focus and was not

similar in appearance to Z-rings that were seen at the midcell later in the cell cycle. Polar foci were also seen in
induced cultures, but were harder to quantify because of
the increase in the overall fluorescence signal as a result
of the higher concentration of FtsZ in these cells (Figs 1A
and 2E and F); however, the proportion of cells with polar
foci was essentially the same in induced cultures as in
uninduced cultures (data not shown). Polar foci were seen
in 56% of cells at 0 min and had decreased to 14% by
60 min (Fig. 3). After 60 min, polar foci were only seen
in a minority of cells. To ensure that the polar localization
of FtsZ early in the cell cycle was not caused by
the presence of pUJ142ftsZ, we quantified the polar
localization of FtsZ in a synchronized population of wildtype strain NA1000 (Fig. 4). Polar localization was
observed at a similar frequency in the wild-type NA1000
population except at 0 min, when polar FtsZ was seen in
24% of cells compared with 56% in NA1000/pUJ142ftsZ.
Polar FtsZ increased to 51% at 15 min and had
decreased to 11% by 60 min. The rapid rise and decrease
in polar FtsZ indicates that FtsZ resides at this location for
only a brief period at the beginning of the cell cycle.
Polar FtsZ could result from residual FtsZ at the
previous site of cell division. To address this possibility,
cells of a synchronized population of NA1000 were
Fig. 2. Immunolocalization of FtsZ in induced
and uninduced cultures at different times in
the cell cycle. Overlaid images of phase and
FtsZ immunofluorescence (white) in a
synchronized culture are shown for the times
indicated in minutes above and below for
uninduced (G, glucose) and induced (X,
xylose) cultures.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

952 E. M. Quardokus, N. Din and Y. V. Brun

at 0 min, and as this proportion increased between 43%
and 51% from 15 to 45 min, these results indicate that
most of the polar FtsZ seen early in the cell cycle cannot
be a remnant of the previous cell division.
We used immunogold transmission electron microscopy to examine the polar localization of FtsZ more
precisely. Cells from a synchronized population of wildtype strain NA1000 were fixed, embedded in resin and
thin sectioned. The thin sections were incubated with antiFtsZ antibody, washed and incubated with colloidal goldconjugated goat anti-rabbit secondary antibody. The
background signal in the absence of primary antibody

Fig. 3. FtsZ localization and cell constriction during the cell cycle.
FtsZ was localized by immunofluorescence, and the number of
cells with a specific pattern of localization is indicated as a
percentage of cells examined. A minimum of 200 cells was counted
for each time point. For Z-rings, we counted cells with two dots
opposite one another and bands of FtsZ staining as in Fig. 2FI. Zrings were quantified in NA1000/pUJ142ftsZ, in which the synthesis
of FtsZ was either uninduced (A) or induced (W). For cell
constrictions, we counted cells with any sign of constriction visible
on images captured with a CCD camera on a phase-contrast
microscope with a 100 objective. Cells with one constriction were
quantified in NA1000/pUJ142ftsZ, in which the synthesis of FtsZ
was either uninduced (B) or induced (X), and cells with two
constrictions were quantified in NA1000/pUJ142ftsZ, in which the
synthesis of FtsZ was induced (D). Representative FtsZ
immunolocalization pictures are shown for the different stages of
the uninduced cell cycle. The cell cycle is depicted
diagrammatically, and the DNA replication period is shown at the
bottom.

analysed for FtsZ localization by immunofluorescence

immediately after cell division (180 min). Polar FtsZ was
seen at the previous site of cell division (the pole opposite
the stalk) in 11% of stalked cells, suggesting that these
polar FtsZ foci were remnants of the previous cell division
(Table 1). In swarmer cells, polar FtsZ was seen in 14% of
cells, but the nature of the pole was not established
(Table 1). As synchronized swarmer cells had approximately the same proportion of cells with polar FtsZ, 24%

Fig. 4. FtsZ localization in wild-type NA1000 cells throughout the

cell cycle.
A. In situ immunofluorescence localization of FtsZ in synchronized
cells. The time in the cell cycle is indicated for each part. The arrow
at 15 min points to a cell with polar FtsZ.
B. Graph of polar (V) and mid-cell (W) localization of FtsZ.
C. Immunoblot of equal volumes of cells at different stages in the
cell cycle indicated in minutes. At 150 min, swarmer (SW) and
stalked (ST) cells were separated, and an amount of cells
equivalent to the amount at the start of the synchrony was
analysed.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

Regulation of FtsZ ring formation in Caulobacter

Table 1. FtsZ localization immediately after cell division.
Percentage of cells with localization pattern
Localization pattern

Swarmer

Mid-cell
0
Stalked pole

Opposite stalked pole

Either pole
14
No localization
86

Stalked

Predivisional

15
7
11

67

100

a
Cells of a synchronized population of NA1000 were analysed for
FtsZ localization by immunofluorescence microscopy at the time of
cell division (180 min). At least 100 cells of each type were counted.

and with preimmune serum as the primary antibody was

negligible. Representative examples of gold labelling are
shown in Fig. 5. Clusters of gold particles could be
seen at the ends of swarmer cells in the early stages of
the cell cycle (Fig. 5A). In addition, cells that were
sectioned through the stalk at early stages of stalk
synthesis had gold clusters associated with the incipient
stalk (Fig. 5BD). Gold labelling was also observed in the
cytoplasm of cells at early stages of the cell cycle, but
cytoplasmic labelling was much lower than polar labelling.
Quantification of the number of gold particles at polar
versus non-polar locations indicated that cells at early
stages of the cell cycle had an average of 629 gold
particles mm22 at the pole and 34 gold particles mm22 in
the rest of the cell. These results confirm the polar
localization of FtsZ detected by immunofluorescence and
indicate that FtsZ is located at the incipient stalked pole of
the cell early in the cell cycle.

953

sufficient to form a Z-ring (Figs 1 and 3). Z-rings could not

be detected until cells had passed the 30 min stage in the
cell cycle in both induced and uninduced cultures (Fig. 3).
The Z-rings began to form slightly earlier in induced
cultures than in uninduced cultures, although Z-ring
formation was negligible until 45 min into the cell cycle.
This time corresponds to the differentiation of swarmer
cells into stalked cells and coincides with the initiation
of DNA replication. To ensure that the timing of mid-cell
FtsZ localization was not caused by the presence of
pUJ142ftsZ, we quantified the percentage of cells containing mid-cell FtsZ during the cell cycle of a synchronized population of wild-type strain NA1000. Again, FtsZ
mid-cell localization began to be detected after swarmer
cell differentiation as the percentage of polar FtsZ
decreased (Fig. 4). We conclude that Z-rings cannot
form in swarmer cells, but that they can form very soon
after swarmer cell differentiation.
Random cytoplasmic foci represent the third main
pattern of localization observed (Fig. 2E). These random
foci were abundant in induced cultures (Fig. 2). Very few
random foci were seen in the uninduced culture or in wildtype NA1000 cells. This suggests that the random

Z-ring formation occurs early in the cell cycle

After the polar FtsZ staining had disappeared in most cells
in the uninduced and induced population, mid-cell FtsZ
staining began to appear. This second staining pattern
was observed predominantly after 90 min into the cell
cycle. Most cells had fluorescent foci opposite one
another at mid-cell (Fig. 3, fourth cell picture) or fluorescent bands at mid-cell (Fig. 2JL, NP and Fig. 3, cell
pictures 57). We interpreted these signals as indicative
of Z-rings as described previously (Addinall and Lutkenhaus, 1996). A single fluorescent focus could occasionally
be seen at the mid-cell of unconstricted cells. This
localization pattern may represent the beginning of Zring formation at a nucleation site (Fig. 3, third cell) and
was also observed in wild-type cells (data not shown).
We quantified the number of cells containing either FtsZ
bands or two foci of FtsZ staining opposite each other at
mid-cell as indicative of Z-rings (Fig. 3). These data
indicate that Z-rings do not form in swarmer cells, even in
induced cultures in which the concentration of FtsZ is
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

Fig. 5. Polar localization of FtsZ by immunogold transmission

electron microscopy. Electron micrographs of fixed and sectioned
cells from a synchronized population of NA1000 treated with
affinity-purified anti-FtsZ antibody and visualized using goat antirabbit 12 nm colloidal gold conjugates as the secondary antibody.
Polar clusters of gold particles are indicated by arrows. Cells were
collected at 0 min (C), 30 min (A) and 90 min (B and D).
Bars 200 nm.

954 E. M. Quardokus, N. Din and Y. V. Brun

Fig. 6. Morphology of cells uninduced and induced for FtsZ overexpression.

AH. Phase-contrast micrographs. Aliquots of cells from synchronized populations of Caulobacter cells carrying pUJ142ftsZ, in which FtsZ
overexpression was either uninduced (AD) or induced (EH), were removed and visualized by light microscopy. The various parts are
representative of cellular morphology at 105 min (A and E), 135 min (B and F), 165 min (C and G) and 195 min (D and H).
IN. Transmission electron micrographs of cells induced for FtsZ overexpression. Aliquots of cells induced for FtsZ overexpression were
removed at different times after cell synchronization, fixed and examined by transmission electron microscopy.
I. A cell from the uninduced culture at 150 min into the cell cycle.
JL. Cells induced for FtsZ expression at 150 min into the cell cycle.
M and N. Cells at around 300 min into the cell cycle.
I, J and K. The bars represent 500 nm.
L, M and N. The bars represent 1 mm.

cytoplasmic foci are the result of polymerization or

aggregation of excess FtsZ in the induced culture.
FtsZ overexpression does not affect the timing of cell
constriction
To determine the effect of FtsZ overproduction on the timing
of cell constriction, we synchronized swarmer cells from
cultures in which FtsZ expression was either uninduced or
induced and quantified the number of cells with visible
constrictions at 15 min time points throughout the cell cycle.
At each time point, aliquots of cells were fixed, and the cells
were examined by phase-contrast microscopy.
The synchronized culture in which FtsZ expression was
not induced progressed normally through the cell cycle;
stalks became visible at around 45 min (data not shown).
At 75 min, only 4% of the cells had begun to constrict. The
first clear signs of constriction (. 10% of cells) occurred
at 90 min into the cell cycle when 13% of cells had begun
to constrict (Fig. 3). At the 105 min time point, < 30% of
cells had constrictions, as shown in Fig. 6A. At 135 min,
the proportion of cells with visible constrictions reached a
maximum at 74%. By 180 min, most of the cells had
completed cell division, and the number of cells with
constrictions had decreased to 34%.
The synchronized culture in which FtsZ expression was

induced did not exhibit morphological differences at the

beginning of the cell cycle compared with the uninduced
culture (data not shown); stalks became visible at 45 min
(data not shown). At 75 min, 9% of cells had begun to
constrict (Fig. 3). At approximately 105 min, 32% of the
cells had visible mid-cell constrictions, which was only
slightly more than the 30% observed in the uninduced
culture. By 165 min, 80% of cells had a single constriction. The majority of cells in the induced culture did not
complete cell division efficiently (Fig. 6H); cell separation
was delayed, and a second constriction began to appear
around 135 min after the start of the cell cycle (Figs 3 and
6F). By 165 min, 16% of cells had two constrictions
(Figs 3 and 6G), and the total number of cells with one or
two constrictions was 96% (Fig. 3). Cell separation
eventually occurred, and approximately 45% of the cells
had separated 210 min from the start of the cell cycle.
When the percentage of constricted cells was plotted
versus time in the cell cycle, the curves for the uninduced
and induced cultures were almost overlapping (Fig. 3).
The graph in Fig. 3 indicates that, in the uninduced
culture, the Z-rings appeared 30 min before the first signs
of constriction. In the induced culture, the Z-rings
appeared < 45 min before cell constriction occurred.
The addition of xylose to cells containing pUJ142 without
an insert did not affect cell division (not shown). Thus,
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

Regulation of FtsZ ring formation in Caulobacter

even when the concentration of FtsZ was sufficient in
swarmer cells for cell division to occur, Z-ring formation
and cell constriction could not occur earlier in the cell
cycle. Furthermore, there was a delay of at least 30 min
between these two events. We conclude that FtsZ
concentration is not sufficient for Z-ring formation and
the initiation of cell division.
Spatial restriction of second Z-ring formation in
FtsZ-overproducing cells
The fact that FtsZ overexpression delayed cell separation
allowed us to determine whether there were any restrictions in the positioning of additional constrictions. The
second constriction of cells in which FtsZ overexpression
was induced was always positioned close to the initial
constriction site and was never observed at the cell ends
(Fig. 6). When the second constriction began to form, the
two constrictions were separated from one another by
< 0.6 mm and were < 1.5 mm from each cell end (Fig. 6J
and K). Cells with two constrictions often continued to
grow and to form new constrictions, yielding cells with
many constrictions (Fig. 6L and N). These new constrictions also occurred close to a previous constriction.
Eventually, these multiply constricted cells divided,
resulting in cells with constrictions near one pole and
with either a stalk or a flagellum at the other pole.
We used immunofluorescence to determine whether the
placement of a second constriction was caused by a
restriction in Z-ring localization. We stained for FtsZ in a
synchronized induced culture grown in PYE medium,
because FtsZ staining produced less random foci than
when grown in M2-G. This is probably a result of the fact
that induction of FtsZ synthesis in NA1000/pUJ142ftsZ is
stronger in M2G than in PYE (Fig. 1A). The doubling time
of cells in PYE is approximately half that in M2-G. At
90 min, the majority of the cells had a single area of FtsZ
staining at the mid-cell, suggesting the presence of a single
Z-ring. In contrast, cells taken at 135 min had a wider FtsZ
staining area that was often composed of two distinct
staining areas (data not shown). DNA staining was often
seen between the two FtsZ staining areas. We conclude
that these cells have two distinct Z-rings. A few cells (data
not shown) had four distinct FtsZ staining areas, suggesting the presence of four Z-rings. These results indicate that
a mechanism exists to confine cell constriction to the midcell region in cells overexpressing FtsZ.
Depletion of FtsZ occurs in both swarmer and stalked
cells at cell division
We have shown previously that, when swarmer and
stalked cells were separated after cell division, only the
stalked cell fraction contained a detectable amount of
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

955

FtsZ by immunoblotting (Quardokus et al., 1996; Kelly

et al., 1998). The possibility remained that the FtsZ
detected in the stalked cell fraction resulted from the few
predivisional cells that are present in the stalked cell
fraction (Gober et al., 1991). This is supported by
immunofluorescence analysis of FtsZ in a synchronized
population of wild-type NA1000 cells immediately after
cell division (180 min). In this population, mid-cell FtsZ
localization was only detected in 15% of newborn stalked
cells, whereas it was detected in 100% of the remaining
predivisional cells (Table 1). In fact, the majority of
swarmer and stalked cells, 86% and 67%, respectively,
had no FtsZ localization (Table 1). Immunoblot analysis of
FtsZ in equal amounts of cells taken at various times
during the cell cycle and in separated swarmer and
stalked cell fractions immediately after cell division
(150 min) indicated that most of the FtsZ present during
the cell cycle was degraded at cell division (Fig. 4C).
Densitometry scanning of the immunoblot indicated that
the stalked cell fraction contained 25% of the maximum
amount of FtsZ present during the cell cycle (90 min).
Some of the FtsZ in the stalked fraction was a result of the
contaminating predivisional cells. In addition, when the
immunoblot was overexposed, a faint FtsZ band could be
seen in the swarmer cell fraction. Quantification of
swarmer cell FtsZ by densitometry revealed that the
swarmer cell fraction contained < 10% of the amount of
FtsZ present in the stalked fraction, which is equivalent to
2.5% of the FtsZ level at its maximum. Taken together,
these data indicate that at least 75% of the FtsZ present
at the time of cell division initiation is degraded at cell
division.

Discussion
An important advantage of using Caulobacter to study cell
division is that it can easily be synchronized to yield a
population of swarmer cells that are unable to replicate
their chromosome. DNA replication is initiated upon
differentiation of swarmer cells into stalked cells, and
chromosome replication occurs once and only once
during each cell cycle (Marczynski, 1999). This makes it
possible to correlate each step during the progression of
cell division to a stage of chromosome replication. The
synthesis of FtsZ begins during swarmer cell differentiation (Quardokus et al., 1996). The results of our
experiments show that synthesizing FtsZ in swarmer
cells induces Z-ring formation slightly earlier than in wildtype cells; however, no Z-rings could be detected in
swarmer cells even when they contained a concentration
of FtsZ sufficient to form a Z-ring. This provides evidence
that the developmental cell cycle dictates the timing of
Z-ring formation and that the concentration of FtsZ is not

956 E. M. Quardokus, N. Din and Y. V. Brun

the only factor driving Z-ring assembly and the initiation of
cell division.
We propose three main models to explain the inability
of FtsZ to form Z-rings in swarmer cells. First, FtsZ
may be unable to polymerize into a stable Z-ring because
inhibitors of polymerization are present. Our immunofluorescence data for swarmer cells that contain FtsZ
argue, in part, against the possibility that FtsZ polymerization is completely inhibited in swarmer cells. The
polar localization and the random fluorescent foci seen in
swarmer cells in these experiments presumably require
the polymerization of at least a few FtsZ molecules to be
visible in the fluorescence microscope. It appears that
FtsZ is able to form at least short polymers in swarmer
cells, although a caveat to this argument is that the
random fluorescent foci could result from aggregation of
FtsZ molecules. The second model suggests that FtsZ is
unable to form a stable Z-ring because a target is absent.
Perhaps the interaction of FtsZ with the putative target or
nucleation site is under cell cycle control and only occurs
in stalked cells.
Our third model is that Z-ring formation is coupled to an
early stage in DNA replication by nucleoid occlusion or
some other mechanism. The nucleoid occlusion model
states that all sites along cells can serve as division sites.
The nucleoids are thought to block Z-ring formation at all
sites, except at the mid-cell once the nucleoids have
segregated (Mulder and Woldringh, 1989; Woldringh et al.,
1990; Yu and Margolin, 1999). Although swarmer cell
nucleoids occupy the whole cell and could inhibit Z-ring
formation everywhere, the coincidence of Z-ring formation
with swarmer cell differentiation indicates that very little
DNA replication and segregation of the nucleoids is
required for Z-ring formation. In fact, when FtsZ was
overproduced, additional mid-cell constrictions appeared
3040 min after the time required for the completion of
DNA replication. This is equivalent to the duration of the
post-synthetic gap in DNA replication (Degnen and
Newton, 1972) and suggests that the ability to reinitiate
cell division is coupled to DNA replication. For example,
the movement of one of the newly replicated origins to the
pole of the cell immediately after the initiation of DNA
replication (Jensen and Shapiro, 1999) could provide a
signal for Z-ring formation. This would be consistent with
the fact that the chromosome segregation genes parA
and parB are essential for cell viability (Mohl and Gober,
1997). Recent experiments indicate that an early stage of
DNA replication is required for Z-ring formation (E. M.
Quardokus and Y. V. Brun, in preparation). In Bacillus
subtilis, Z-rings can form even if DNA replication is
blocked early in germinating spores, but most of the
Z-rings form at non-central positions under these conditions (Harry et al., 1999).
The coupling of Z-ring formation to an early stage of

DNA replication in Caulobacter provides one checkpoint

of many that couple cell division to DNA replication In
addition, progression of cell division after the initiation of
constriction requires ftsA and ftsQ whose transcription
late in the cell cycle is dependent on DNA replication,
providing a later checkpoint (Wortinger et al., 2000).
Caulobacter mutants in the topoisomerase IV parC and
parE genes produce filamentous cells that are highly
pinched at multiple sites, indicating that cell separation
requires chromosome decatenation (Ward and Newton,
1997).
Our results indicate that the concentration of FtsZ is not
the only factor driving the initiation of cell constriction,
which begins < 30 min after Z-ring formation. The time
required for cell constriction to begin corresponds
approximately to the time required to replicate half of
the chromosome, as determined by a chromosome
methylation assay (Stephens et al., 1996). This indicates
that cell constriction starts well before the termination of
chromosome replication. Thus, the timing of Z-ring
formation and cell constriction relative to chromosome
replication in Caulobacter is quite different from that in
E. coli, in which Z-ring formation occurs near the
termination of DNA replication (Den Blaauwen et al.,
1999). The delay between Z-ring formation and cell
constriction could simply reflect the time required between
localizing FtsZ at the mid-cell and the formation of a
complete and functional Z-ring or cell division complex.
For example, the appropriately timed expression of FtsW,
a protein required for constriction to begin in E. coli (Boyle
et al., 1997; Khattar et al., 1997), may be necessary to
initiate cell constriction.
Overproduction of FtsZ in Caulobacter caused the
formation of additional constrictions that were confined to
the mid-cell. This is true even after many cell cycles, as
we have never observed divisions at the stalked pole
when FtsZ is overproduced. The inability of Caulobacter
poles to serve as constriction sites contrasts with the
situation in E. coli, in which a slight overproduction of
FtsZ (two- to sevenfold) causes divisions at the cell
poles to produce minicells (Ward and Lutkenhaus, 1985).
Under normal conditions in E. coli, the Min proteins
prevent Z-ring formation at non-mid-cell positions
(Rothfield et al., 1999); however, minCDE homologues
are absent in Caulobacter (http://www.tigr.org/tdb/mdb/
mdbinprogress.html). Immunofluorescence microscopy
has indicated that, when FtsZ was overproduced, the
formation of Z-rings was confined to a small area close to
the initial Z-ring at the mid-cell. The inability of FtsZ to
form rings at the poles of the cell is certainly not the result
of a complete inhibition of FtsZ localization at the poles,
as cells early in the cell cycle contain some FtsZ localized
at a pole of the cell. Polar FtsZ could have a specific
function early in the cell cycle. The localization of FtsZ at
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

Regulation of FtsZ ring formation in Caulobacter

the incipient stalked pole and the timing of its localization
suggests the intriguing possibility that FtsZ is required for
the initiation of stalk synthesis, as suggested previously
(Brun et al., 1994; Quardokus et al., 1996). Perhaps FtsZ
marks this pole as a target for the localization of stalk
synthesis proteins. Experiments are in progress to test
this hypothesis formally.
Finally, we have shown that FtsZ is almost completely
depleted from both swarmer and stalked cells at cell
division. These results are consistent with the hypothesis
that FtsZ stability is controlled by its assembly state (Kelly
et al., 1998). Our previous work using only immunoblot
analysis had led to the hypothesis that FtsZ was much
more stable in the stalked compartment of predivisional
cells than in the swarmer compartment at the time of cell
division (Kelly et al., 1998). The combination of immunoblot and immunofluorescence analysis presented here
indicates that this hypothesis is most likely wrong and that
FtsZ is degraded rapidly in both cell compartments at cell
division. It is still possible that the protease that degrades
FtsZ is inactivated when FtsZ synthesis resumes early in
the cell cycle.

957

Purification of FtsZ-His, antibody production and

immunodetection
A 69 kDa recombinant FtsZ protein with a C-terminal sixhistidine tag was expressed from BL21lDE3/pftsZCN13 after
1 mM IPTG induction overnight. The insoluble protein was
collected, solubilized from inclusion bodies and purified over
a nickel-charged HIS-Bind resin column as described by the
manufacturer (Novagen) under 6 M urea denaturing conditions. The resulting protein was used as an antigen for
antibody production. Polyclonal antibodies were raised in
New Zealand white rabbits at CoCalico Biologicals. Crude
antiserum was affinity purified over an AmidoLink Plus
coupling gel column (Pierce) to which FtsZ-His protein was
covalently bound, or as described previously (Din et al.,
1998). Affinity-purified anti-FtsZCC primary antibody was
used for immunomicroscopy and immunoblot analysis. For
immunoblot analysis, cell extracts were resolved by SDS
PAGE and transferred to nitrocellulose (Schleicher and
Schuell). FtsZ was detected using either crude antiserum
containing polyclonal antibodies raised against full-length
FtsZ used at a 1:6000 dilution or affinity-purified antibodies at
a 1:1000 dilution. The secondary antibody, a goat anti-rabbit
IgG (H1L)horseradish peroxidase (HRP) conjugate (Gibco
BRL), was preadsorbed with C. crescentus NA1000 acetone
powders (Maddock and Shapiro, 1993) and used at a dilution
of 1:20 000. Immunoblots were quantified using a Molecular
Dynamics densitometer with IMAGEQUANT software.

Experimental procedures
Bacterial strains, plasmids, media and growth conditions

In situ immunofluorescence labelling and microscopy

E. coli DH5alphaF 0 (Liss, 1987) was used as the host for

cloning, S17-1 (Simon et al., 1983) was used for conjugation,
and BL21lDE3 was used to overproduce FtsZ-His for
antibody production. The C. crescentus NA1000 strain is a
synchronizable derivative of strain CB15 (Evinger and
Agabian, 1977). All C. crescentus and E. coli strains were
grown as described previously (Brun and Shapiro, 1992). The
construction of plasmid pUJ142ftsZ, carrying full-length FtsZ,
has been described previously (Din et al., 1998).

In situ immunofluorescence labelling of Caulobacter cells was

performed using a modification of the procedure described by
Den Blaauwen et al. (1999). Briefly, cells were fixed in 2.5%
formaldehyde, 30 mM sodium phosphate buffer, pH 7.5, for
15 min to 2 h at room temperature. Fixed cells were spun at
7000 r.p.m. for 8 min in a microcentrifuge and washed twice
in 1 phosphate-buffered saline (PBS), pH 7.2. To permeabilize them, fixed cells were incubated in 0.1% Triton X-100 in
PBS for 45 min at room temperature. Cells were washed
three times as before and resuspended in 1 PBS containing
5 mM EDTA and incubated for 5 min at room temperature (to
preserve the stalk structure) before adding 100 mg ml21
lysozyme and incubating for an additional 45 min at room
temperature. Cells were washed three times and resuspended in 0.5% (w/v) blocking reagent from Roche Molecular
Biochemicals (catalogue no. 1096176) in 1 PBS for 30 min
at 378C. Primary antibody (affinity-purified anti-FtsZCC) was
diluted in blocking reagent for 60 min at 378C. Cells were
washed three times in 1 PBS. Secondary antibody
[fluorescein isothiocyanate (FITC)-conjugated goat antirabbit; Jackson Immunological Research] was diluted in
blocking buffer for 30 min at 378C. Cells were washed three
times in PBS20.05% Tween 20. DNA was stained with
propidium iodide at a concentration of 10 mg ml21. Cells were
washed once in 1 PBS and resuspended in SlowFade
(Molecular Probes). Cells were observed as described
previously (Wortinger et al., 1998). For phase-contrast
microscopy, cells were removed from cultures and fixed in
an equal volume of 3.4% formaldehyde. Light microscopy
was performed on a Nikon Eclipse E800 light microscope

DNA manipulations, genetic techniques and cell

synchronization
Genetic techniques were carried out as described previously
(Brun and Shapiro, 1992). Swarmer cells were isolated
from exponential cultures grown in M2-G (Johnson and Ely,
1977), M2-GX or PYE (Poindexter, 1964) medium and
processed as described previously (Evinger and Agabian,
1977). M2-GX is M2-G containing 0.3% (w/v) xylose.
pET21b1 (Novagen) was used to construct an overexpression clone of the full-length C. crescentus ftsZ gene. The
resulting pftsZCN13 was transformed into BL21lDE3 for overexpression. The following primers were used to amplify the fulllength ftsZ: FtsZN (5 0 -AAAAAACATA TGGCTATTTCTCTTTC
CGCGCCG-3 0 ) replaces the start codon of ftsZ with an NdeI site,
and FtsZCH (5 0 -AAAAAACGAATTCGCGTTGGCCAGGCGGC
GCAGGAACGA-3 0 ) replaces the ftsZ stop codon with an
EcoRI site.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

958 E. M. Quardokus, N. Din and Y. V. Brun

with a 100 Plan Apo oil objective. Images were captured
using a Princeton Instruments Cooled CCD camera model
1317 and the METAMORPH imaging software package version
4.1 (Universal Imaging Corporation).

Quantification of FtsZ during the cell cycle

A synchronized population of swarmer cells isolated from
strain NA1000 cells was allowed to proceed through the cell
cycle. Aliquots of cells were removed at the given times
for SDSPAGE and immunoblot analysis, and the number
of molecules of FtsZ present at different stages of the cell
cycle was determined by comparing the density of the FtsZ
protein bands with known amounts of FtsZ-His run on the
same gel.

Immunogold transmission electron microscopy

Samples for immunotransmission electron microscopy were
prepared as described previously (Alley et al., 1992), except
that glutaraldehyde was omitted from the fixing medium, and
Tween 20 was omitted from blocking, antibody incubation
and washing buffers. Cells were embedded in LR White,
sectioned and placed on 0.25% Formvar-dipped 200-mesh
nickel grids (Ted Pella) as described previously (Wright and
Rine, 1989). Sections were incubated with undiluted affinitypurified antibody and washed in 1 PBS. Colloidal gold
(12 nm)-conjugated goat anti-rabbit secondary antibody
(Jackson Immunological Research) was used at a 1:50
dilution in 2% BSA in 1 PBS. Grids were counterstained
with 7.5% uranyl magnesium acetate for 1 min, followed by
three washes in sterile filtered deionized water. Grids were
observed using a JEOL-JEM-1010 transmission electron
microscope at 60 kV. Negatives were scanned using a
UMAX flatbed scanner and Adobe Photoshop software.
Area measurements were performed using NIH IMAGE 1.6.1
software (developed at the US National Institutes of
Health and available on the Internet at http://rsb.info.nih.
gov/nih-image/), and the number of gold particles was
counted and expressed as particles per area (mg2). Background signal observed with secondary antibody alone was
01 gold particles cell21 and with preimmune serum was
02 gold particles cell21.

Acknowledgements
We thank members of our laboratory for critical reading of the
manuscript, and Rudi Turner for sectioning embedded
samples. This work was supported by National Institutes of
Health grant GM51986 to Y.V.B.

References
Addinall, S.G., and Lutkenhaus, J. (1996) FtsZ-spirals and
-arcs determine the shape of the invaginating septa in
some mutants of Escherichia coli. Mol Microbiol 22:
231237.
Alley, M.R.K., Maddock, J.R., and Shapiro, L. (1992) Polar

localization of a bacterial chemoreceptor. Genes Dev 6:

825836.
Bi, E., and Lutkenhaus, J. (1990) FtsZ regulates frequency
of cell division in Escherichia coli. J Bacteriol 172: 2765
2768.
Bi, E.F., and Lutkenhaus, J. (1991) FtsZ ring structure
associated with division in Escherichia coli (see comments). Nature 354: 161164.
Boyle, D.S., Khattar, M.M., Addinall, S.G., Lutkenhaus, J.,
and Donachie, W.D. (1997) ftsW is an essential celldivision gene in Escherichia coli. Mol Microbiol 24: 1263
1273.
Brun, Y.V., and Janakiraman, R. (2000) The dimorphic life
cycle of Caulobacter and stalked bacteria. In Prokaryotic
Development. Brun, Y.V., and Shimkets, L.J. (eds).
Washington, DC: American Society for Microbiology
Press, pp. 297317.
Brun, Y.V., and Shapiro, L. (1992) A temporally controlled
sigma factor is required for cell-cycle dependent polar
morphogenesis in Caulobacter. Genes Dev 6: 23952408.
Brun, Y., Marczynski, G., and Shapiro, L. (1994) The
expression of asymmetry during cell differentiation. Annu
Rev Biochem 63: 419450.
Dai, K., and Lutkenhaus, J. (1992) The proper ratio of FtsZ to
FtsA is required for cell division to occur in Escherichia coli.
J Bacteriol 174: 61456151.
Degnen, S.T., and Newton, A. (1972) Chromosome replication during development in Caulobacter crescentus. J
Mol Biol 64: 671680.
Den Blaauwen, T., Buddelmeijer, N., Aarsman, M.E.,
Hameete, C.M., and Nanninga, N. (1999) Timing of FtsZ
assembly in Escherichia coli. J Bacteriol 181: 51675175.
Din, N., Quardokus, E.M., Sackett, M.J., and Brun, Y.V.
(1998) Dominant C-terminal deletions of FtsZ that affect its
ability to localize in Caulobacter and its interaction with
FtsA. Mol Microbiol 27: 10511064.
Evinger, M., and Agabian, N. (1977) Envelope-associated
nucleoid from Caulobacter crescentus stalked and swarmer
cells. J Bacteriol 132: 294301.
Garrido, T., Sanchez, M., Palacios, P., Aldea, M., and
Vincente, M. (1993) Transcription of ftsZ oscillates during
the cell cycle of Escherichia coli. EMBO J 12: 39573965.
Gober, J.W., Champer, R., Reuter, S., and Shapiro, L. (1991)
Expression of positional information during cell differentiation in Caulobacter. Cell 64: 381391.
Harry, E.J., Rodwell, J., and Wake, R.G. (1999) Coordinating DNA replication with cell division in bacteria: a
link between the early stages of a round of replication and
mid-cell Z ring assembly. Mol Microbiol 33: 3340.
Jensen, R.B., and Shapiro, L. (1999) The Caulobacter
crescentus smc gene is required for cell cycle progression
and chromosome segregation. Proc Natl Acad Sci USA 96:
1066110666.
Johnson, R.C., and Ely, B. (1977) Isolation of spontaneously
derived mutants of Caulobacter crescentus. Genetics 86:
2532.
Kelly, A.J., Sackett, M.J., Din, N., Quardokus, E., and Brun,
Y.V. (1998) Cell cycle-dependent transcriptional and
proteolytic regulation of FtsZ in Caulobacter. Genes Dev
12: 880893.
Khattar, M.M., Addinall, S.G., Stedul, K.H., Boyle, D.S.,
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

Regulation of FtsZ ring formation in Caulobacter

Lutkenhaus, J., and Donachie, W.D. (1997) Two polypeptide products of the Escherichia coli cell division gene
ftsW and a possible role for FtsW in FtsZ function. J
Bacteriol 179: 784793.
Liss, L.R. (1987) New M13 host: DH5aF 0 competent cells.
Focus 9: 13.
Lu, C., Stricker, J., and Erickson, H.P. (1998) FtsZ from
Escherichia coli, Azotobacter vinelandii, and Thermotoga
maritima quantitation, GTP hydrolysis, and assembly.
Cell Motility Cytoskeleton 40: 7186.
Lutkenhaus, J., and Addinall, S.G. (1997) Bacterial cell
division and the Z ring. Annu Rev Biochem 66: 93116.
Maddock, J.R., and Shapiro, L. (1993) Polar location of the
chemoreceptor complex in the Escherichia coli cell.
Science 259: 17171723.
Marczynski, G.T. (1999) Chromosome methylation and
measurement of faithful, once and only once per cell
cycle chromosome replication in Caulobacter crescentus. J
Bacteriol 181: 19841993.
Meisenzahl, A.C., Shapiro, L., and Jenal, U. (1997) Isolation
and characterization of a xylose-dependent promoter from
Caulobacter crescentus. J Bacteriol 179: 592600.
Mohl, D.A., and Gober, J.W. (1997) Cell cycle-dependent
polar localization of chromosome partitioning proteins in
Caulobacter crescentus. Cell 88: 675684.
Mulder, E., and Woldringh, C.L. (1989) Actively replicating
nucleoids influence positioning of division sites in Escherichia coli filaments forming cells lacking DNA. J Bacteriol
171: 43034314.
Ohta, N., and Newton, A. (1996) Signal transduction in the
cell cycle regulation of Caulobacter differentiation. Trends
Microbiol 4: 326332.
Ohta, N., Grebe, T.W., and Newton, A. (2000) Signal
transduction and cell cycle checkpoints in developmental
regulation of Caulobacter. In Prokaryotic Development.
Brun, Y.V., and Shimkets, L.J. (eds). Washington, DC:
American Society for Microbiology Press, pp. 341359.
Palacios, P., Vicente, M., and Sanchez, M. (1996) Dependency of Escherichia coli cell-division size, and independency of nucleoid segregation on the mode and level
of ftsZ expression. Mol Microbiol 20: 10931098.
Poindexter, J.S. (1964) Biological properties and classification of the Caulobacter group. Bacteriol Rev 28:
231295.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 949959

959

Quardokus, E., Din, N., and Brun, Y.V. (1996) Cell cycle
regulation and cell type-specific localization of the FtsZ
division initiation protein in Caulobacter. Proc Natl Acad Sci
USA 93: 63146319.
Rothfield, L., Justice, S., and Garcia-Lara, J. (1999) Bacterial
cell division. Annu Rev Genet 33: 423448.
Sackett, M.J., Kelly, A.J., and Brun, Y.V. (1998) Ordered
expression of ftsQA and ftsZ during the Caulobacter
crescentus cell cycle. Mol Microbiol 28: 421434.
Simon, R., Prieffer, U., and Puhler, A. (1983) A broad
host range mobilization system for in vivo genetic
engineering: transposon mutagenesis in gram-negative
bacteria. Biotechnology 1: 784790.
Stephens, C., Reisenauer, A., Wright, R., and Shapiro, L.
(1996) A cell cycle-regulated bacterial DNA methyltransferase is essential for viability. Proc Natl Acad Sci USA 93:
12101214.
Ward, J.E., Jr, and Lutkenhaus, J. (1985) Overproduction of
FtsZ induces minicell formation in E. coli. Cell 42: 941949.
Ward, D., and Newton, A. (1997) Requirement of topoisomerase IV parC and parE genes for cell cycle progression and
developmental regulation in Caulobacter crescentus. Mol
Microbiol 26: 897910.
Woldringh, C.L., Mulder, E., Valkenburg, J.A., Wientjes, F.B.,
Zaritsky, A., and Nanninga, N. (1990) Role of the nucleoid
in the toporegulation of division. Res Microbiol 141: 3949.
Wortinger, M.A., Quardokus, E.M., and Brun, Y.V. (1998)
Morphological adaptation and inhibition of cell division
during stationary phase in Caulobacter crescentus. Mol
Microbiol 29: 963973.
Wortinger, M., Sackett, M.J., and Brun, Y.V. (2000) CtrA
mediates a DNA replication checkpoint that prevents cell
division in Caulobacter crescentus. EMBO J 19: 4503
4512.
Wright, R., and Rine, J. (1989) Transmission electron
microscopy and immunocytochemical studies of yeast:
analysis of HMG-CoA reductase overproduction by
electron microscopy. Methods Cell Biol 31: 473512.
Yu, X.C., and Margolin, W. (1999) FtsZ ring clusters in min
and partition mutants: role of both the Min system and the
nucleoid in regulating FtsZ ring localization. Mol Microbiol
32: 315326.