Download The chromosome partitioning protein, ParB, is required for

Molecular Microbiology (2001) 42(3), 741–755
The chromosome partitioning protein, ParB, is required
for cytokinesis in Caulobacter crescentus
Dane A. Mohl,† Jesse Easter Jr and
James W. Gober*
Department of Chemistry and Biochemistry, and the
Molecular Biology Institute, University of California,
Los Angeles, CA 90095 –156, USA.
Summary
In Caulobacter crescentus, the genes encoding the
chromosome partitioning proteins, ParA and ParB,
are essential. Depletion of ParB resulted in smooth
filamentous cells in which DNA replication continued.
Immunofluorescence microscopy revealed that the
formation of FtsZ rings at the mid-cell, the earliest
molecular event in the initiation of bacterial cell
division, was blocked in cells lacking ParB. ParB
binds sequences near the C. crescentus origin of
replication. Cell cycle experiments show that the
formation of bipolarly localized ParB foci, and
presumably localization of the origin of replication
to the cell poles, preceded the formation of FtsZ
rings at the mid-cell by 20 min. These results suggest
that one major function of ParA and ParB may
be to regulate the initiation of cytokinesis in
C. crescentus.
Introduction
The bacterial cell cycle is a deceptively simple process,
which, for most organisms, culminates in cell division
when the pre-divisional cell reaches a critical size of twice
the unit mass. Therefore, newly divided rod-shaped
bacteria, such as Escherichia coli, are remarkably similar
in size, usually varying no more than 10 per cent in length.
As in eukaryotic cells, the bacterial cell cycle has been
divided into periods of DNA synthesis, chromosome
partitioning or segregation and, finally, cell division. The
timing of each of these processes must be highly
coordinated to ensure that each daughter cell inherits a
full complement of genetic material. However, in contrast
to eukaryotic cells, the mechanisms in bacteria that serve
Accepted 26 July, 2001. *For correspondence. E-mail gober@
chem.ucla.edu; Tel. (11) 310 206 9449; Fax (11) 310 206 5213.
†
Present address: Department of Biology, California Institute of
Technology, Pasadena, CA, USA.
Q 2001 Blackwell Science Ltd
to couple DNA replication, partitioning and cell division are
largely unknown.
For most bacteria, replication initiation follows the
binding of the replication initiator protein, DnaA to the
replication origin (reviewed in Kornberg and Baker, 1992).
Although the regulation of initiation varies widely in
different bacteria, one aspect typically involves a refractory
period in which DnaA is prevented from binding to the
origin. For example, in E. coli, hemi-methylated sequences
in the origin promote the binding of SeqA that antagonizes
DnaA binding (Lu et al., 1994; Slater et al., 1995). In
Caulobacter crescentus, the response regulator CtrA
fulfils a similar function (Quon et al., 1998). Upon
completion of DNA synthesis, the circular daughter
chromosomes are separated through the action of
topoisomerases and terminus-specific recombinases,
and then partitioned toward the poles of the pre-divisional
cell (reviewed in Rothfield, 1994; Wake and Errington,
1995). Partitioning has been hypothesized to be a
necessary prerequisite for cytokinesis, which, in E. coli
cells, requires a period of post-replication protein
synthesis (Donachie and Begg, 1989). It is not known
whether this period of protein synthesis supplies
proteins for chromosome partitioning or cell division. The
earliest known cytological event in bacterial cytokinesis is
the assembly of FtsZ in a mid-cell ring around the
circumference of the cell. FtsZ is a bacterial homologue of
tubulin and is required for the formation of a constriction at
the site of cell division. The assembly of the FtsZ ring
results in the recruitment of other cell division proteins,
such as FtsA, FtsQ, FtsI and FtsK, to the mid-cell location,
a process that eventually culminates in cell division
(reviewed in Bramhill, 1997).
How is chromosome partitioning linked to cell division?
Until recently, few details of the chromosome-partitioning
process were known. Unlike eukaryotic cells, bacteria lack
a defined mitotic spindle or other cytoskeletal structures
that might be involved in directing the newly replicated
chromosomes toward the poles of the cell. It is envisioned
that the partitioning apparatus must consist of elements
that are analogous to those involved in chromosome
segregation in eukaryotic cells: a centromere binding
protein, a mechanism to move the chromosomes towards
the cell poles and, possibly, a mechanism of condensing
the nascent chromosomes. Recent experiments have
shown that ParA and ParB, the cellular homologues of
742 D. A. Mohl, J. Easter, Jr. and J. W. Gober
partitioning proteins found in some phage and unit-copy
plasmids, may have a critical role in partitioning bacterial
chromosomes (Ireton et al., 1994; Sharpe and Errington,
1996; Glaser et al., 1997; Lin et al., 1997; Mohl and Gober,
1997).
Although the genes encoding these proteins are found in
virtually all bacterial genomes sequenced thus far (the
notable exception being enteric bacteria and Haemophilus
influenzae ), most of our information regarding their
function comes from elegant work on the E. coli
bacteriophage P1, which exists as a circular episome
when a prophage, and the F-factor plasmid. Both of these
genetic elements exist as single-copy episomes and are
inherited by each daughter cell with remarkable efficiency,
even in the absence of selection. It has been demonstrated that mutations at three loci, parA, parB and a
cis-acting site, parS (sopABC respectively, in F-factor),
result in a plasmid-partitioning defect (Austin and
Abeles, 1983; Mori et al., 1986. parB has been shown
to encode a DNA-binding protein that specifically
recognizes the parS sequence that functions as the
plasmid equivalent of a centromere (Davis and Austin,
1988; Mori et al., 1989). ParA is an ATPase, whose
activity is stimulated by the presence of ParB bound to
DNA (Davis and Austin, 1988). These proteins are
hypothesized to interact with host factors, resulting in
plasmid segregation. This idea is supported by the
cytological observation that newly replicated F-factor
plasmids move towards the poles of the cell only in the
presence of wild-type copies of sopA and sopB (Niki and
Hiraga, 1999).
An analogous role has been formulated for the cellular
homologues of ParA and ParB. In the sporulating
bacterium, Bacillus subtilis, the parB homologue, spo0J,
is required for entry into the sporulation pathway. Strains
containing mutations in spo0J accumulate anucleate cells
(up to 3%) under vegetative growth conditions, indicative
of a possible role in chromosome partitioning (Ireton et al.,
1994). The block in sporulation in spo0J mutants is
attributable to a lack of Spo0A-regulated transcriptional
activation (Ireton et al., 1994; Cervin et al., 1998; Quisel
et al., 1999). Spo0A is a global regulator of early sporulation genes whose activity is controlled by phosphorylation
in response to several internal and external stimuli.
Interestingly, mutations in the parA homologue, soj
(spo0JA ) relieve the sporulation requirement for Spo0J,
suggesting that these two proteins may function as components of a switch that regulates Spo0A phosphorylation
in response to chromosome partitioning (Ireton et al.,
1994).
In the dimorphic bacterium, C. crescentus, parA and
parB are located in an operon that maps near the origin of
replication (Cori). In contrast to B. subtilis, both parA and
parB are essential for viability (Mohl and Gober, 1997).
Cell cycle expression experiments have shown that
although the transcription of parAB varies during the cell
cycle, the cellular levels of each protein remain constant
throughout the cell cycle, suggesting that the synthesis of
stoichiometric amounts of each protein is critical for
function. In support of this idea, overexpression of either
parA or parB leads to a significant defect in chromosome
partitioning (Mohl and Gober, 1997). Cell cycle immunolocalization experiments have shown that the subcellular
distribution of ParB parallels that of chromosome movement; initially, there exists a single focus of ParB, localized
to one pole of the cell, and as the cell cycle progresses,
another focus is localized to the opposite pole. Presumably, the bipolar localization of ParB reflects binding to
the origin of replication region, as fluorescent in situ
hybridization (FISH) experiments have shown that the
C. crescentus origin region is localized to the poles of the
pre-divisional cell (Jensen and Shapiro, 1999). In
unsynchronized cultures, ParA was also shown to be
bipolarly localized in pre-divisional cells (Mohl and Gober,
1997). Similar results have been obtained for Spo0J
localization in B. subtilis (Glaser et al., 1997; Lin et al.,
1997). Caulobacter crescentus and B. subtilis ParB
homologues have been shown to bind to DNA sequences
adjacent to the origin of replication (Mohl and Gober, 1997;
Lin and Grossman, 1998). Furthermore, the origin of
replication in B. subtilis has been visualized during the
cell cycle and shown to segregate rapidly towards the
poles of the cell (Webb et al., 1997; 1998). These
observations in C. crescentus and B. subtilis, as well as
experiments that monitored sporangium-specific gene
expression in spo0J mutants (Sharp and Errington, 1996),
have led to the hypothesis that ParB (Spo0J), and possibly
ParA (Soj), function to position the origin of replication region
of the chromosome towards the poles of the pre-divisional
cell as the initial event in chromosome partitioning.
In this report, we investigate the molecular nature of the
lethality of parB null mutations. We demonstrate that
strains that are depleted of ParB undergo a block in cell
division with the accumulation of smooth filamentous cells
in culture. Furthermore, we demonstrate that this block in
cell division is attributable to a defect in FtsZ ring
assembly, the earliest known event in bacterial cytokinesis. Cell cycle immunolocalization experiments show that
the formation of bipolar foci of ParB precedes FtsZ ring
formation. As previous experiments have demonstrated
that ParA and ParB are synthesized in stoichiometric
amounts during the cell cycle, an increase in the ratio of
ParA to ParB may be resulting in a cell division block. We
tested whether overexpression of ParA would also result in
a cell division defect. Overexpression of ParA led to the
production of filamentous that lacked FtsZ rings, however,
simultaneous overexpression of both proteins had no such
cell division defect.
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ParB is required for cell division in Caulobacter
Results
Depletion of ParB blocks cell division and FtsZ ring
formation
To investigate the lethality of parB null mutations, we
constructed a strain in which the only copy of parB was
under control of a promoter inducible by xylose (xylAp ).
This strain (UC9031) was unable to form colonies on
complex media lacking xylose, confirming previous results
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indicating that parB is an essential gene. These cells were
grown overnight in media containing 8 mM xylose and then
inoculated into fresh medium lacking xylose. Then, we
assayed the relative levels of ParA and ParB, as well as
monitoring the phenotype, over time, using phase contrast
microscopy and immunofluorescent microscopy (Fig. 1).
The levels of ParB decreased to an almost undetectable
concentration by 8 h following the removal of xylose from
the medium, whereas the ParA concentration remained
Fig. 1. Depletion of ParB results in a cell division block that produces long smooth filaments. Caulobacter crescentus strain UC9031 possesses a
frame-shifted copy of parB at the par operon locus, and a wild-type copy of the parB gene in the xylose-inducible xylA operon. To eliminate parB
expression, log phase cultures were washed and then suspended in PYE medium without xylose. Samples were removed every 2 h for immunoblot
(A and B), DAPI staining and FtsZ immunofluorescence (C).
A. Immunoblot showing the relative levels of ParA and ParB under conditions of ParB depletion. The time in hours (hrs) following depletion is
indicated. ParA and ParB were detected using specific antiserum.
B. Quantitative representation of the immunoblot data shown in A. ParA levels are represented by circles. ParB levels are represented by squares.
C. DAPI staining (left and right panels) and FtsZ immunofluorescence (right panels) in ParB-depleted cells. All panels are combined phase and
fluorescence micrographs. Between 0 and 4 h, most cells exhibit normal morphology and DNA staining. By 6 h, cells are continually elongating but
not dividing. By 8 h, depletion of ParB produces long filaments: very few cells possess constrictions associated with division sites and most filaments
are smooth. No defect in DNA staining is apparent, suggesting that replication is not adversely effected by ParB depletion or cell filamentation. FtsZ
ring formation was visualized with polyclonal anti-FtsZ antibody (right panels) and goat anti-rabbit IgG antibody conjugated to Cy3. FtsZ rings appear
as bright orange bands at unpinched division sites, and then as spots at pinched sites. Between 0 and 4 h, ParB-depleted cells demonstrated
normal patterns of division and FtsZ ring formation. White arrows identify FtsZ rings near the mid-cell. After 6 h, cells began to divide less frequently
but some were still competent for FtsZ ring formation. After 8 h, ParB-depleted cells were elongated and the frequency of FtsZ ring formation is
decreasing dramatically. In addition, some polar divisions resulted in FtsZ ring formation near the terminus of the cell. Almost all cells are elongated
filaments, and FtsZ rings are absent following 10 h of depletion. See Fig. 2A for a quantitative representation of this experiment.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 741–755
744 D. A. Mohl, J. Easter, Jr. and J. W. Gober
Fig. 2. Depletion of ParB from Caulobacter crescentus blocks
assembly of new FtsZ rings and cell division.
A. The frequency of FtsZ ring staining between 0 and 10 h after ParB
depletion. Multiple fields of each time-point were counted. At time 0 h,
38% of cells possess FtsZ rings (total of 382 cells counted). After 2
and 4 h, 52% and 43% of cells possessed rings (197 and 246 cells
counted respectively). A sharp decline in FtsZ ring formation occurred
between 4 and 6 h. The ratios of FtsZ ring staining at 6, 8 and 10 h,
were 15% (244 cells counted), 10% (81 cells counted) and 7% (113
cells counted) respectively.
B. Cell perimeter length was estimated from phase micrographs of
multiple fields. Images were captured and analysed with NIH IMAGE
software. Values are relative units of perimeter length. After ParB
depletion for 4 – 6 h, Caulobacter cells began to elongate. The lack of
FtsZ ring formation points towards a block early in the cell division
process. Growth rate is not adversely effected by ParB depletion as
cell mass rather than cell number continues to double. However,
viability decreased to 0.072%, following 10 h of ParB depletion.
relatively constant (Fig. 1A and B). Following 4–6 h of
ParB depletion, a slight defect in cell growth was readily
observed (Figs 1C and 2A). The first notable effect was an
aberrant placement of the site of cell division, with the
formation of anucleate minicells and larger cells containing
DNA (data not shown). Over time, there was an
accumulation of long, smooth filamentous cells in culture
(Figs 1C and 2A). This suggests that the depletion of ParB
from these cells results in a cell division block. Wild-type
cells, when shifted from xylose-containing medium to a
medium that contained only glucose, showed no such cell
division block (data not shown). Interestingly, many of
the cell division events that did occur took place at nonmid-cell locations, very often near the pole. The presence
of cell division events at non-mid-cell or polar locations has
also been observed when C. crescentus cells are depleted
of the replication initiation protein, DnaA (Gorbatyuk and
Marczynski, 2001). The simplest interpretation of the
experiment presented here is that ParB is required for cell
division, and thus provides an explanation for the lethality
of parB null mutations. This is in stark contrast to mutations
that result in partitioning defects in other organisms such
as mukB mutations in E. coli (Hiraga et al., 1989) and
spo0J mutations in B. subtilis that are not lethal (Ireton
et al., 1994).
The accumulation of smooth filaments, when the cells
were depleted of ParB, indicated that cell division was
blocked at an early stage. As conditional ftsZ mutants in
E. coli form smooth filaments, we tested whether there
was a defect in FtsZ localization under conditions of ParB
depletion. The assembly of a ring of FtsZ, that girdles the
circumference of the cell at a mid-cell location, is the
earliest known cytological event in the initiation of cell
division (reviewed in Bramhill, 1997). When strain UC9031
was grown in media containing xylose, and then shifted to
medium without xylose for 2 h, there was a fraction of cells
that exhibited a band of FtsZ localization at the mid-cell, as
assayed by immunofluorescence microscopy (Fig. 1C).
This result is consistent with previous observations
showing that FtsZ rings are present in only a fraction of
pre-divisional cells, as localization occurs at a specific time
in the cell cycle (Quardokus et al., 2001). As ParB was
depleted from these cells, there was a gradual accumulation of cells that did not possess FtsZ rings. In those cells
that did have rings, they were often located near the poles,
an expected finding as some of the cell division events
under these conditions produce minicells. The simplest
interpretation of this result is that a lack of ParB blocks
FtsZ ring formation.
The smooth morphology of ParB-depleted cells
suggested that they were blocked at an early stage of
cell division. To determine if depletion of ParB affects a
step in cell division, before or following formation of FtsZ
rings, we monitored the rate of FtsZ ring formation and the
rate of cell growth during the course of ParB depletion. If
ParB acts at a step in cell division that follows FtsZ ring
assembly, the fraction of elongated cells with intact FtsZ
rings should remain relatively constant during the initial
stages of filamentation; if ParB is necessary for the
assembly of new FtsZ rings, then we should observe a
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ParB is required for cell division in Caulobacter
decrease in the frequency of FtsZ rings that is concurrent
to cell elongation. Our results indicate that the latter model
is correct, as FtsZ ring-containing cells decreased as a
fraction of the population at the same time-points that
filamentation appeared to begin (Fig. 2B). Therefore, ParB
is required for either the synthesis of FtsZ or the assembly
of FtsZ rings, but not for division at previously existing
sites.
Mechanism of the inhibition of FtsZ ring formation
What mechanism is operating under these conditions to
block FtsZ ring formation? One formal possibility is that
ParB depletion activates an inhibitor of cell division.
Several inhibitors of FtsZ ring formation have been
described in E. coli. The most relevant amongst these is
the SOS-inducible protein, SulA. The SOS response is
characterized by the induction of several genes involved
in DNA repair and an inhibition of cell division in response
to either DNA damage or an inhibition of DNA replication
(reviewed in Walker, 1996). Under these conditions,
SulA prevents the polymerization of FtsZ. Although
C. crescentus undergoes a typical SOS response when
treated with DNA damaging agents (J. C. Draper and J. W.
Gober, unpublished), it is not known what protein inhibits
cell division under these conditions. Analysis of the
C. crescentus genome sequence, as well as other
completed microbial genomes, has revealed that the
occurrence of sulA is restricted to enteric bacteria. As the
identity of the SOS-regulated cell division inhibitor is
unknown, we used an indirect assay for an SOS response.
We reasoned that the most probable pathway for induction
of an SOS response under these conditions would be an
inhibition of DNA replication. To test this possibility, we
assayed replication by measuring the incorporation of
[a-32P]-dGTP into DNA (Marczynski et al., 1990) during
the course of ParB depletion. We found that the rate of
DNA replication in ParB-depleted cells, even after 10 h of
depletion, was not significantly less than control cells in
which ParB was not depleted (Table 1). Furthermore, flow
cytometry experiments demonstrated that ParB-depleted
cells contained a range of six to 14 chromosomes (data not
shown). Although this is a indirect test, we conclude from
this experiment that the block in FtsZ ring formation in
ParB-depleted cells is probably not attributable to an SOS
response. To provide an additional test for SOS induction,
we assayed the rate of transcription of the C. crescentus
uvrA promoter that is induced by an SOS response
(J. C. Draper and J. W. Gober, unpublished observation).
Depletion of ParB resulted in less than a twofold increase
in uvrA transcription, a result consistent with a lack of SOS
induction (data not shown).
In E. coli, both FtsZ and FtsA are required for the
formation of a stable ring structure. One possibility is that
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745
Table 1. The effect of ParB depletion on the rate of DNA replicationa
Time (h)
0
2
4
6
8
cpm/OD
83 147
59 058
97 645
78 524
89 088
a. The rate of DNA replication was determined by pulse labelling cells
with [a-32P]-dGTP (Marczynski et al., 1990) and is expressed as
scintillation counts per minute [cpm/OD (A660nm)].
the synthesis of these two components is inhibited in ParBdepleted Caulobacter cells. To test this possibility, we
measured the expression of lacZ from both the ftsZ and
ftsQA promoters in normal and ParB-depleted cultures
(Fig. 3). Interestingly, depletion did not block transcription
from either reporter. Instead, we observed a small
increase in expression from both the ftsZ and ftsQA
promoter constructs. To ensure that the transcription of the
ftsZ promoter resulted in the synthesis of FtsZ, we used
polyclonal antibodies directed towards FtsZ to investigate
the relative levels of protein in wild-type and ParB-depleted
cells. We detected an approximate 50% decrease in FtsZ
protein levels by Western blot analysis of ParB-depleted
UC9031 grown in medium without xylose. In light of the
Fig. 3. Transcription does not decrease from either ftsZ or ftsQA
promoters when Caulobacter is depleted of ParB.
A. b-Galactosidase activity generated from ftsZ (placZ/290HB2.0 BP )
and ftsQA (pMSP8LC) promoter fusions to a reporter lacZ in ParB
depletion strain UC9031. Cells were grown either in the presence of
xylose, ParB-inducing conditions, or the absence of xylose, ParB
depletion conditions. b-Galactosidase activity increased slightly from
both promoters when Caulobacter cells were depleted of ParB for 8 h.
ftsZ promoter activity increased from 5900 to 7400 Miller units. ftsQA
promoter activity increases from 5000 to 6900 Miller units.
B. FtsZ protein levels in Caulobacter strain UC9031 were determined
by immunoblot on cultures grown in either the presence (1) or
absence ( – ) of xylose. FtsZ levels appeared to decrease in ParBdepleted cultures.
746 D. A. Mohl, J. Easter, Jr. and J. W. Gober
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ParB is required for cell division in Caulobacter
increased transcription from the ftsZ and ftsQA promoters,
the observed decrease in protein is possibly because of
the instability of unpolymerized FtsZ. In Caulobacter, FtsZ
protein also undergoes cell cycle proteolytic degradation
(Kelly et al., 1998). Overall, these results suggest that the
expression of ftsZ and ftsQA is not significantly affected by
ParB depletion. These experiments do not rule out the
possibility that either ParA or ParB regulate other, as yet
unidentified, cell division promoters, although it may be
that they interact directly with the division apparatus.
We have shown previously that a change in the relative
levels of ParA or ParB results in a cell division defect. In
the depletion experiments presented here, the ratio of
ParA to ParB would also be greatly increased. Therefore,
we tested whether overexpression of ParA would result in
the same defect in FtsZ ring formation and growth as cells
that were depleted of ParB (Fig. 4). In this experiment, an
extra copy of either parA, or both parA and parB, was
placed under control of the xylA promoter in wild-type cells.
The phenotype of the cells was monitored microscopically
over time. Overexpression of parA resulted in a phenotype
that was virtually indistinguishable from ParB-depleted
cells (Fig. 4, A–F). There was a relatively rapid inhibition of
normal cell division and FtsZ ring formation, with
filamentous cells and some minicells appearing in culture
as little as 6 h after the addition of xylose. These results
indicate that overexpression of parA inhibits FtsZ ring
formation and division. To test whether raising ParB levels
can suppress the effect of ParA overexpression, we
placed both parA and parB under control of the xylA
promoter. In this case, cell division was restored, but the
majority of cells in culture possessed abnormal morphology. Elongated and shortened cells may result from
aberrantly placed division events. This could explain our
previous observation that co-overexpression results in
anucleate cells. Altogether, these results suggest that
ParA may inhibit FtsZ ring formation, and ParB might
serve to regulate ParA activity, such that partitioning and
cell division are coupled.
The formation of a partitioning complex precedes FtsZ ring
formation
The experiments presented above show that ParB is
747
required for recruitment of FtsZ to the site of cell division.
Furthermore, altering the ratio of ParA:ParB also results in
a cell division defect. In previous experiments, we have
demonstrated that overexpression of ParA results in a loss
of ParB foci and that overexpression of ParB causes the
formation of mislocalized ParB foci, often at non-polar
locations (Mohl and Gober, 1997). These results may
indicate that the mislocalization of either ParA or ParB is
responsible for the cell division defect. For wild-type cells,
we propose that the cell cycle localization of ParB and
ParA and, by implication, the origin of replication region to
the poles of the cell, is a required event for FtsZ ring
formation and cell division. To test this idea, we assayed
the cell cycle-dependent localization of ParB and FtsZ in
synchronized populations of cells. Swarmer cells were
isolated (. 95% pure) by density centrifugation, suspended in fresh medium and allowed to progress through
the cell cycle. At various time-points, a portion of the
culture was removed, the cells were fixed and processed
for immunofluorescent microscopy using antibodies
directed against ParB and FtsZ. These experiments
indicated that arrival of ParB foci, to both poles of the
cell, occurs at about 60 min into the cell cycle, shortly after
the swarmer to stalked cell transition, and concurrent with
chromosome replication (Fig. 5). Notably, the arrival of
ParB foci at poles preceded FtsZ ring formation. To
demonstrate better the close relationship between ParB
localization and cell division, we graphed the fraction of
cells with bipolar ParB foci and mid-cell FtsZ ring
structures over time (Fig. 6). The sigmoidal nature of
both curves is indicative of a sharp change in the pattern of
localization during the cell cycle. The data shows that
bipolar ParB localization occurred approximately 20 min
before the formation of FtsZ rings.
ParB stimulates the initiation of FtsZ ring formation and
cell division
Next, we tested whether reintroduction of ParB should
stimulate FtsZ ring formation in ParB-depleted cultures. To
accomplish this, we grew strain UC9031 in medium
without xylose for 6 h, and then added xylose, inducing
expression of parB from the xylA promoter. Cells were
fixed and FtsZ rings were stained by immunofluorescence
Fig. 4. Altered expression of ParA and ParB results in FtsZ ring and cell division defects. Overexpression of ParA from the xylose promoter on a
multicopy plasmid. parA was expressed for 0 – 10 h from a xylose-inducible promoter. Greater than 100 cells were counted at each time-point. The
frequency of FtsZ ring formation dropped from 42% at time 0 (A), to 34% after 2 h of xyl-parA expression (B),and then to 21% after 4 h (C). By 6 h
most cells were elongated and only 13% possessed FtsZ rings (D). After 8 h, 9% of the cells possessed FtsZ rings and at 10 h, only 4% (E and F). In
contrast, cells which had both parA and parB overexpressed together possessed normal FtsZ ring formation. Before induction of xylose-inducible
expression, Caulobacter possessed normal morphology and FtsZ ring staining (G). Following 10 h of expression, some elongated and shortened
cells resulted from asymmetrical divisions (H). FtsZ ring formation was not inhibited, but occurred at inappropriate sites. As reported previously (Mohl
and Gober, 1997), some of these divisions resulted in anucleate cells, suggesting that FtsZ ring formation and division are not co-ordinately timed.
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ParB is required for cell division in Caulobacter
749
normal length (Fig. 7D). In addition, a number of cells
(10%) contained more than one FtsZ ring. As these cells
possess multiple chromosomes, this suggests that the
number of chromosomes in the cell, and, by implication
the number of ParB foci, may influence the frequency of
cell division events in Caulobacter.
Discussion
Fig. 6. Bipolar localization of ParB foci precedes FtsZ ring formation.
The number of cells demonstrating bipolar ParB localization, and
FtsZ ring assembly was determined from fluorescence and phase
images in Fig. 6. The average number of cells demonstrating either
bipolar ParB localization, or FtsZ ring formation, is graphed against
time. The resulting graph demonstrates the arrival of ParB to both
poles approximately 20 min before FtsZ ring formation, suggesting
that duplication and partitioning of ParB foci could serve as a cell
division cycle cue.
during depletion and then after ParB induction (Fig. 7). As
expected, depletion of ParB resulted in a dramatic
decrease in FtsZ ring formation and cell division. After
6 h, cells became long filaments with fewer than 10%
exhibiting FtsZ rings. The induction of ParB expression
caused a dramatic increase in the number of cells with
FtsZ rings, and subsequently the frequency of division.
One hour after inducing ParB expression, over 30% of
cells possessed FtsZ rings. After 2 h, more than 70% of the
culture possessed FtsZ rings. The relatively rapid
recruitment of the cell division apparatus supports the
idea that ParB is acting as a positive regulator of division.
Interestingly, the placement of rings in some filaments did
not follow a pattern that would probably result in cells with
A long-standing problem in prokaryotic biology has been
understanding how bacterial cells regulate progression
through the cell cycle. Newly replicated chromosomes
must be partitioned to opposite poles of the pre-divisional
cell before the completion of division, implying that these
two processes are temporally coupled. Here we present
evidence that ParA and ParB, the cellular homologues of
plasmid-partitioning proteins, are required for a specific
step in the cell division cycle: the onset of cytokinesis. In
previous work, it has been demonstrated that both parA
and parB are required for viability in Caulobacter (Mohl
and Gober, 1997). To investigate the nature of the lethality
of parB mutations, we constructed a Caulobacter mutant in
which the sole copy of parB was placed under the control
of a xylose-inducible promoter. When cultures were
removed from xylose and depleted of ParB, a marked
cell division phenotype appeared. After 4–6 h, the cells
elongated, forming long, smooth filaments. The lack of
constrictions or invaginations of the cell wall suggested
that ParB depletion inhibited an early step in the division
process. Formation of the FtsZ ring, which provides the
scaffolding required for septal constriction at the division
site, is the earliest known molecular event in the initiation
of cell division (reviewed in Bramhill, 1997). To test
whether the cell division defect observed in ParB depletion
mutants was attributable to a block in the initiation of
division, we assayed the formation of FtsZ rings in wildtype and ParB-depleted cultures. Using immunofluorescence microscopy, we found that ParB is essential for the
formation of new FtsZ rings but not necessarily required
Fig. 5. Cell cycle timing of polar ParB localization and FtsZ ring formation suggests that newly replicated origins are partitioned before FtsZ ring
formation. A wild-type culture (LS107) was synchronized by centrifugation through Percoll and released into growth medium. Two portions of cells
were fixed every 20 min and prepared for either ParB or FtsZ immunofluorescence. The time, in minutes, is indicated to the left of the micrographs. In
the left hand column, orange foci of Cy3 fluorescence are localized ParB. In the right hand column, FtsZ immunofluorescence and phase images
were merged. Orange bars across the middle of the cell are Cy3-stained FtsZ rings. These experiments demonstrate that ParB and, therefore, the
two newly replicated origins are localized to both poles of stalked cells before FtsZ rings assemble at the mid-cell. In swarmer and early stalked cells
(0 – 40 min), ParB is localized to only one pole (89% at 0 min; 73% at 20 min; 70% at 40 min), and FtsZ rings have not yet formed (98.5% to 97.2%
without rings at these time-points). White arrows indicate staining at one pole. After 60 min, 61% of stalked cells demonstrated bipolar localization of
ParB and approximately 24% of cells had FtsZ rings. Arrows indicate ParB staining and FtsZ ring formation. In 88% of late stalked cells (80 min),
ParB was localized to both poles, and in 63% of late stalked cells FtsZ rings have formed. After 100 min, greater than 94% of cells possessed bipolar
localization of ParB and 86% had FtsZ rings. The pre-divisional cells began to divide and new ParB foci appeared between 120 and 140 min. In these
cells, two foci of ParB sometimes appeared in the stalked cell compartment of the pre-divisional cell (shown by arrows; ParB column, 140 min).
Between 100 and 500 cells were counted at each time-point. A quantitative representation of this experiment is depicted as Fig. 7.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 741–755
750 D. A. Mohl, J. Easter, Jr. and J. W. Gober
Fig. 7. Induction of parB in depleted cells stimulates FtsZ ring formation and cell division. Depletion of ParB from an otherwise wild-type culture
resulted in the production of long filaments that lack FtsZ rings. To demonstrate the reversible nature of the ParB division block, parB expression was
induced using the xylA promoter in cells after 6 h of depletion. Immunofluorescence was performed on fixed cultures after either depletion or
reintroduction of ParB to assay the reappearance of FtsZ rings.
A. and B. 0 (A) and 2 h (B) after removing strain UC9031 from the presence of xylose, cells still divided normally as ParB levels have not dropped
below a critical threshold concentration. White arrows indicate FtsZ rings at the mid-cell.
C. After 4 h, the cells began to elongate and fewer FtsZ rings were present.
D. After 6 h of depletion, fewer than 10% of cells had FtsZ rings and greater than 90% had become elongated filaments.
E. One hour after inducing ParB expression in the previously depleted culture, approximately 30% of cells possessed one or more FtsZ rings. White
arrows indicate Z rings. Newly formed rings appeared to be randomly distributed along the length of the cell filament.
F. Two hours after induction of ParB, greater than 70% of the culture had formed FtsZ rings. White arrows indicate rings that have formed beside one
another.
Fig. 8. ParA and ParB activities are required for
cytokinesis. Light grey spheres represent ParA,
dark grey spheres are ParB and black line
drawings represent the chromosomes. We
hypothesize that ParA functions as a cell
division inhibitor and ParB regulates its activity
with respect to the completion of DNA
partitioning. ParB bound to the chromosome, is
initially localized to one pole of the swarmer
and early stalk cells. Replication of the origin
region results in the duplication of ParB foci.
One ParB/origin DNA complex remains at the
stalked pole whereas the other is partitioned
to the new swarmer pole. Before replication,
ParA is capable of inhibiting FtsZ ring formation
and is distributed throughout the cell.
Localization of ParB to both poles may
sequester ParA activity away from the mid-cell,
resulting in FtsZ ring assembly and then
division.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 741–755
ParB is required for cell division in Caulobacter
for the completion of division from previously existing
structures. In addition, induction of ParB synthesis in
depleted cells resulted in the reappearance of FtsZ rings.
We hypothesize that the inhibition of cell division,
observed in these cells, is responsible for the lethality of
null mutations in parB. These results are consistent with
the view that ParB is required for the initiation of cell
division.
Fluorescence microscopy experiments have revealed a
simple cycle in which the origin and terminus regions of
bacterial chromosomes become localized to the pole and
mid-cell in a cell cycle-dependent fashion. For example, in
B. subtilis the origins of newly replicated chromosomes are
abruptly moved towards opposite poles during the cell
cycle (Webb et al., 1997; 1998). The observed discontinuous and rapid rate of movement supports so-called
‘active’ models of partitioning. A similar distribution of
origin and terminus DNA in synchronized populations of
C. crescentus has also been observed (Jensen and
Shapiro, 1999). Our hypothesis is that the initiation of cell
division is not only dependent on the synthesis but also
upon the polar localization of ParB bound to parS
sequences near to the origin of replication. Approximately
six ParB DNA-binding sites, almost identical in sequence
to those found in B. subtilis (Lin and Grossman, 1998), are
distributed adjacent to the par operon and the origin of
replication in Caulobacter (J. Easter, D. A. Mohl and J. W.
Gober, unpublished). Arrival of ParB, bound to these DNA
sites, probably signals the duplication and successful
partitioning of the origin of replication, and, thereby,
releases the cell division block. Therefore, ParB recognizing its DNA binding sites could be part of a chromosomecounting mechanism, using the two poles as a means to
distinguish between identical DNA molecules. If polar
localization of ParB is required for division, then arrival of
ParB foci to the poles of the cell should precede the
formation of FtsZ rings. In support of this, we have shown
that the arrival of ParB at the cell poles precedes FtsZ ring
formation by approximately 20 min (Fig. 6). In previous
work, we have determined that ParA also is localized to the
poles of the cell at the pre-divisional stage (Mohl and
Gober, 1997). We envisage that cell division initiates when
ParA interacts with the ParB –DNA complex at the poles
of the cell (Fig. 8). ParA may also be inhibiting cell division
in ParB-depleted cells of C. crescentus. In this regard,
overexpression of ParA results in a cell division phenotype
that is identical to ParB-depleted cells, whereas overexpression of both proteins results in a partitioning defect,
but no evident cell division defect. Therefore, these
proteins must be expressed in stoichiometric amounts for
normal cell division to occur. In this case, overexpression
of ParA, and depletion of ParB, both would lead to an
increase in the ratio of ParA to ParB in the cell. We have
determined that depletion of ParB results in an increase in
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 741–755
751
ParA levels (D. A. Mohl and J. W. Gober, not published).
This is a consequence of increased expression of the par
operon that ParB autoregulates. Therefore, it is possible
that under conditions of ParB depletion, ParA may be
inhibiting cell division (Fig. 8).
The deduced amino acid sequence of parA shows that it
is homologous to a large distinct family of ATPases
(Motellebi-Veshareh et al., 1990; Koonin, 1993). Members
in this family have been shown, in many cases, to interact
with a protein that modulates ATPase activity, such as
ParB. We propose that the modulation of ParA ATPase
activity is a molecular switch that responds to spatial
events in the bacterial cell cycle. The plasmid and phageencoded parA and parB gene products have been the
most intensely studied in this regard. The parA gene
products of plasmid and phage have been shown to bind to
DNA sequences within their own promoter and function to
regulate expression of the parAB operon (Friedman and
Austin, 1988; Hayes et al., 1994; Hirano et al., 1998). This
autoregulation is critical for proper partitioning, as overexpression of parA or parB leads to partitioning defects
(Abeles et al., 1985; Funnell, 1988). Purified ParA
possesses a weak ATPase activity in vitro (Davis et al.,
1992; Watanabe et al., 1992; Davey and Funnell, 1994).
Experiments have shown that both ATP and ADP can be
bound to ParA (Davey and Funnell, 1997). The ATPbound form interacts with the partitioning complex (i.e.
ParB bound to parS ), whereas the ADP-bound form
preferentially stimulates the binding of ParA to its operator
sequences (Bouet and Funnell, 1999). It has been
proposed that ATP and ADP function to switch the
activities of ParA within the cell. ParB regulates the switch
in ParA activity as it can stimulate ATP hydrolysis when
bound to parS.
An analogous switch in ParA activities is probably a
critical aspect in regulating cellular processes in bacteria.
For example, partitioning of the origin-proximal third of the
chromosome must occur before placement of the polar
septum to ensure segregation of DNA into the forespore
during sporulation in B. subtilis. Soj (ParA) and Spo0J
(ParB) are thought to operate this developmental
checkpoint by directly regulating the transcription of key
sporulation genes. Soj has been shown to oscillate from
pole to pole, with this dynamic localization being
dependent on Spo0J (Marston and Errington, 1999; Quisel
et al., 1999) In the absence of Spo0J, Soj moves from the
poles and associates, primarily, with the nucleoid in which
it inhibits the transcription of early sporulation promoters.
This switch in activities is a consequence of interaction
with Spo0J at the pole and is probably attributable to a
change in the nucleotide-bound form of Soj (Quisel et al.,
1999).
Evidence presented here suggests that the interaction
of ParA and ParB in Caulobacter modulates their influence
752 D. A. Mohl, J. Easter, Jr. and J. W. Gober
on the initiation of cell division. We do not know the
molecular nature of the inhibition of cell division in
Caulobacter, however, there exist two equally plausible
possibilities. To determine whether Caulobacter ParA and
ParB may be acting at the level of transcription, we
assayed the expression of ftsZ and ftsQA promoters in the
ParB depletion strain. Both appeared to be transcribed at
slightly higher levels when ParB was depleted. Despite the
increase in ftsZ and ftsQA promoter activity, FtsZ protein
levels appear to decrease somewhat (Fig. 4). Given that
the initiation of division and selection of the division site is
not yet fully understood, it is difficult to rule out
transcriptional repression as a mode of ParA inhibition.
Alternatively, ParA may directly inhibit the assembly of the
cell division machinery.
The data presented here raise the question of whether
ParA and ParB function both as partitioning proteins as
well as regulators of cell division. In B. subtilis, direct
experiments that visualized the subcellular localization of
the replication origin indicated no evidence of mislocalization in spo0J mutants (Webb et al., 1997b). This result
suggests that other proteins are involved in orienting the
chromosome. In E. coli, which does not possess Par
protein homologues, the origin of replication is also
oriented towards the cell poles (Gordon et al., 1997; Niki
and Hiraga, 1998). The uniform distribution of DNA in
ParB-depleted filamentous cells indicates that partitioning
is uninterrupted in the absence of ParB in Caulobacter, a
finding that is in agreement with origin-labelling experiments in B. subtilis (Webb et al., 1997). Based on these
observations, we hypothesize that ParA and ParB in
Caulobacter probably function as regulators of cell division
and are not directly involved in the mechanics of
partitioning. The partitioning defects observed when
these proteins are overexpressed (Mohl and Gober,
1997) are probably attributable to perturbations in coordinating cell cycle events.
Experimental procedures
Strains and plasmids
All strains were derived from Caulobacter crescentus
LS107 that is an ampicillin-sensitive, synchronizable strain
(Stephens et al., 1997). Cultures were grown in 0.2% peptone
and 0.1% yeast extract (PYE) (Poindexter, 1964) and
supplemented with antibiotics as indicated. ParB depletion
strain UC9031 was derived from LS107. To create a xyloseinducible parB expression strain, we cloned parB into plasmid
pSNX228 –1 (M. R. K. Alley, unpublished) and integrated a
copy into the chromosome at the xylA locus. Using a two-step
gene replacement strategy, we swapped the wild-type parB
gene at the par locus for a frame-shifted mutant. To create a
frame-shift in parB, we used site-directed mutagenesis
(Kunkel and Roberts, 1987) to insert two basepairs (bp)
after the seventh codon, resulting in a translation stop at
codon 131. Then, this was subcloned into plasmid pDELI3
using endogenous HindIII and Eco RI sites, upstream of parA
and downstream of parB, to create pPBSD1, integrated into
LS107 through homologous recombination, and then transduced into the xylAp –parB expression strain using bacteriophage fCR30. We plated the recipient Caulobacter cells
onto PYE medium containing 2% sucrose and 8 mM xylose
to select for cells that lost the pDELI3-encoded sacB.
Sucrose-insensitive mutants were screened for growth on
media lacking xylose. Those colonies which required xylose
for growth proved to have a frame-shifted copy of parB at
the par locus, resulting in a strain in which the only copy of
parB was under control of the inducible xylA promoter
(UC9031). ParA and ParA/ParB overexpressing strains were
described in an earlier report (Mohl and Gober, 1997).
Reporter strains were created by mating pMSP8LC ( ftsQAlacZ ) (Sackett et al., 1998) and plac290HB2.0BP ( ftsZ-lacZ )
(Kelly et al., 1998) into UC9031, using E. coli S17-1 (Simon
et al., 1983).
Immunofluorescence microscopy
The preparation of anti-ParB antibodies was described
previously (Mohl and Gober, 1997). To prepare anti-FtsZ
antibodies, the Caulobacter ftsZ gene was amplified by
polymerase chain reaction (PCR) using a primer that
introduced a Bam H1 restriction site, just before the start
codon. This was subcloned into the overexpression vector,
pET15b, and histidine-tagged FtsZ was purified using a
nickel-sepharose column under denaturing conditions
according to the manufacturer (Novagen). Antibodies were
prepared by a commercial source (Cocalico). For immunofluorescence microscopy, Caulobacter cultures were grown
to mid-log phase and then fixed with formaldehyde and
glutaraldehyde at final concentrations of 2.5% formaldehyde, 30 mM NaPO4, pH 7.5 and between 0 and 0.03%
glutaraldehyde. Cultures were fixed for 15 min at 308C in a
rotary shaker and for an additional 45 – 60 min on ice. Cells
were then washed three times with phosphate-buffered
saline (PBS) containing 140 mM NaCl, 3 mM KCl, 8 mM
Na2HPO4 and 1.5 mM KH2PO4 and resuspended in 50 mM
glucose, 10 mM EDTA and 20 mM Tris-HCl at pH 7.5. To the
fixed cells, lysozyme was added to a final concentration of
300 mg ml21; 20 ml of cells was placed onto poly-L -lysinetreated slides for 30 s, aspirated until dry, and then rinsed
with approximately 20 ml of distilled water. Slides were then
blocked with PBS containing 0.05% TritonX-100 (PBST)
with 2% bovine serum albumin (BSA) for 15 min and
incubated with either polyclonal rabbit anti-ParB, anti-ParA
or anti-FtsZ antibody. Slides were washed for 20 min in
PBST, then blocked with PBST 1 2% BSA, incubated for 1 h
with Cy3 conjugated mouse anti-rabbit IgG antibody and
diluted 1:1000 in PBST 1 2% BSA (Jackson Biological).
Slides were washed for 20 min in PBST, rinsed with distilled
water, air-dried and then covered with 50% glycerol
containing 0.1 mg ml21 4,6-diamidino-2-phenylindole (DAPI)
and a coverslip. Fluorescent images were captured with an
Optronics cooled CCD camera attached to a Zeiss Axioplan
microscope at 1000 magnification. All images were
captured with a PHOTOSHOP AV capture plug in on a
Macintosh Power PC AV computer.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 42, 741–755
ParB is required for cell division in Caulobacter
753
Microscopy and cell measurements
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Acknowledgements
We thank E. Quardokus for helpful hints regarding immunofluorescence microscopy, Y. Brun for providing reporter
plasmids, and A. Reisenauer and L. Shapiro for assistance
in performing flow cytometry analysis. G. C. Draper purified
C. crescentus FtsZ for antibody production. We are also
grateful to members of our laboratory for helpful comments
on the manuscript. This work was supported by grants
from the National Science Foundation (MCB-9513222 and
MCB-9986127) to JWG.
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