Department of Biochemistry, University of
Sydney, New South Wales 2006, Australia
Using immunofluorescence microscopy, we have examined the
dependency of localization among three Bacillus subtilis
division proteins, FtsZ, DivIB, and DivIC, to the
division site. DivIC is required for DivIB
localization. However, DivIC localization is dependent on
DivIB only at high growth temperatures, at which DivIB is essential for division. FtsZ localization is
required for septal recruitment of DivIB and DivIC,
but FtsZ can be recruited independently of DivIB. These
localization studies suggest a more specific role for DivIB
in division, involving interaction with DivIC.
 |
TEXT |
It is very likely that a large
protein complex forms at the division site in bacteria (20, 23,
25). To elucidate the molecular mechanism of cell division, an
understanding of how this complex forms and functions is necessary. In
Escherichia coli, morphogenetic evidence suggests that the
various division proteins are required at different stages of
septation: for example, FtsZ and FtsW act early, while FtsA, FtsQ, and
FtsI are required for septal elongation and closure (21).
Consistent with the order of action of division proteins, FtsZ
localizes first, then FtsA and ZipA localize independently of each
other (2, 10, 19), and the remaining membrane-bound proteins
assemble in the following order: FtsK (26), FtsQ
(6), FtsL (9), PBP 3 (FtsI) (28), and
FtsN (1). It is unclear precisely when FtsW localizes in
this sequence. In these localization dependency studies, no two
division proteins were interdependent for septal recruitment, strongly
suggesting that in E. coli, proteins are recruited to the
division site in a strict linear sequence (28).
In Bacillus subtilis there is no ZipA or FtsN homolog. FtsZ
and three membrane-bound division proteins, DivIB (FtsQ
homolog) (11), DivIC (FtsL-like) (14,
16), and PBP 2B (PBP 3 homolog) (29), have been shown
to localize to the division site (7, 13, 14, 17, 27). All of
these proteins appear to be required early in septation (5, 7, 8,
12, 16), although PBP 2B is additionally required for the
duration of septal ingrowth (7). As in E. coli,
FtsZ probably assembles early since Z rings can form at midcell in
the absence of detectable localization of FtsL, DivIC,
DivIB, or PBP 2B at 37°C (7, 8, 17, 18). However, the septal recruitment pathway for the membrane-bound division
proteins in B. subtilis appears to be different from that in
E. coli. FtsL depletion at 37°C results in rapid
degradation of DivIC, while DivIB and PBP 2B
localizations are undetectable, suggesting that all three proteins
localize either after FtsL or together with FtsL (7, 8).
Such division protein instability in the absence of another division
protein has not been demonstrated in E. coli, suggesting
that the septation process, or at least the assembly of the division
complex, is regulated somewhat differently in the two organisms.
Surprisingly, when PBP 2B is depleted at 37°C, DivIB and
DivIC localizations are significantly decreased and occur
only at midfilament positions, probably at incomplete septa
(7). In contrast, in E. coli the PBP 2B homolog,
PBP 3, appears to be required only for the recruitment of FtsN
(1). PBP 2B also fails to localize in divIB and
divIC temperature-sensitive mutants at the nonpermissive
temperature, 48°C (7). The interdependency of
DivIB, DivIC, and PBP 2B localization suggests that
these three proteins localize together. However, an alternative
possibility is that at a lower temperature (30°C) PBP 2B localization
assembles earlier than that of DivIB and DivIC, but
at 48°C the latter two proteins are needed to maintain PBP 2B at the
septal site.
To further elucidate how division proteins of B. subtilis
are incorporated into the septal complex, we examined for the first time the dependency of localization of DivIB and
DivIC on each other and the dependency of DivIB and
DivIC localization on FtsZ. Our observations suggest a role
for DivIB in cell division, involving interaction with DivIC.
The B. subtilis strains used are derivatives of B. subtilis 168, which is designated SU5 in our collection. Tryptose
blood agar plates containing appropriate antibiotics were used for
colony growth. Isopropyl-
-D-thiogalactopyranoside
(IPTG) was added to 1 mM, and chloramphenicol, phleomycin, and
spectinomycin were used at 5, 2, and 100 µg/ml,
respectively. Growth in liquid medium was performed in L broth
(without glucose) (22). FtsZ depletion in BB11 cells
(Pspac-ftsZ) (4) was achieved by growth (in
IPTG-containing medium) at 37°C to an A590 of
0.4 and then resuspension in IPTG-lacking medium to an
A590 of 0.1, after filtering and washing with L
broth (in IPTG-lacking medium). The resuspended culture was incubated for 1 h at 37°C and then diluted fourfold in IPTG-lacking medium and grown for a further 30 min at 37°C. Samples for
immunofluorescence microscopy (IFM) and Western blot analysis were
taken prior to and 20, 40, 60, and 90 min after resuspension.
Mid-exponential-phase cultures of the divIB null strain,
SU321 (13), and its congenic parent, SU5, were obtained by
growth at 30°C to an A590 of 0.4. Aliquots
were then diluted eightfold into fresh prewarmed media and shifted to
49°C for 1 h. Samples for IFM and Western blotting were
harvested prior to and 20, 40, and 60 min after the shift. The
temperature-sensitive divIC mutant strain, SU391
(Pspac-RRC), and its parent strain, SU392
(Pspac-divIC) (15), were grown at 30°C to an
A590 of 0.4. Aliquots of both cultures were
filtered, washed in IPTG-lacking medium, resuspended in medium lacking
or containing IPTG to an A590 of ~0.07, and
then incubated at 30 or 45°C for 45 min. Samples of each culture were
collected for IFM and Western blot analysis just prior to and 45 min
after resuspension. DivIB, DivIC, and FtsZ IFM was
performed as described previously (8), with the
following minor modifications. Treatment of microscope slides
with poly-L-lysine before and after the addition of
lysozyme-treated cells to the slide was essential for DivIB
and DivIC detection in SU391 and SU392. For FtsZ detection,
slides were treated with poly-L-lysine prior to the
addition of lysozyme-treated cells to the slide. The cells for
DivIC IFM were, in some cases, incubated in 2% bovine
serum albumin-0.05% Tween 20-5% whole goat serum (Jackson
Immunoresearch) prior to incubation with anti-DivIC antibody. Affinity-purified anti-DivIB, -DivIC, and -FtsZ
antibodies were used at 1/10, 1/50 to 1/200, and 1/400 dilutions,
respectively. Microscopy and image analysis have been described
previously (13). For each experiment, more than 100 cells or
approximately 50 filaments (long nonseptate cells) were scored for the
presence or absence of localizations. For Western blot analysis,
samples (10 ml) of cells were collected at the times listed above and
cell lysates were prepared as described previously (12).
Western blotting and immunodetection were performed using an alkaline
phosphatase detection system (13, 14). Rabbit
anti-DivIB, -DivIC, and -FtsZ antisera were used at
dilutions of 1/500, 1/1,000, and 1/4,000, respectively.
Z rings are required for targeting of DivIB and
DivIC to the division site.
Although it is likely that
Z-ring assembly is required for the recruitment of the membrane-bound
division proteins DivIB and DivIC, this has not
been demonstrated in B. subtilis. To test the effect of FtsZ
depletion on targeting of DivIB and DivIC, strain
BB11 (Pspac-ftsZ) (4) was grown in the presence
of IPTG and then resuspended in IPTG-lacking medium for up to 90 min. Localization of DivIB, DivIC, and FtsZ was examined
by IFM. Prior to resuspension, 19, 29, and 86% of cells contained
DivIB, DivIC, and FtsZ localizations, respectively.
Average cell length began to increase approximately 20 min following
resuspension in IPTG-lacking medium. By 90 min following resuspension,
the mean cell length was 31.9 µm and very few Z rings were observed
(9% of the t0 level). The number of both
DivIB and DivIC localizations also decreased in a
similar manner, with 5 and 6% of the t0 level
present at 90 min, respectively. In control experiments when strain
BB11 was resuspended in IPTG-containing medium, the frequency of septal localizations remained high for all three proteins (50 to 140% of the
t0 level).
The inability of DivIB and DivIC to target to the
septum in FtsZ-depleted cells was not a consequence of decreased levels of these proteins. Western blot analysis showed that DivIB
levels remained unaltered under all conditions described above (not
shown). There appeared to be a slight drop in DivIC signal 20 min after resuspension in either IPTG-containing or IPTG-lacking medium (Fig. 1A). The reason for this is
unknown, but it is not specific to the depletion of FtsZ. No change in
DivIC levels was observed at later times in either medium. As
expected, cellular FtsZ levels dropped with time in the absence of
IPTG, with about a fourfold reduction 90 min after resuspension in
IPTG-lacking medium (not shown). We conclude that although
DivIB and DivIC levels are unchanged in the absence
of Z rings, they are not targeted to the division site, at least at
detectable levels. A previous study showed that when B. subtilis cells are depleted of FtsL, DivIC is degraded, raising the possibility that DivIC is only stable if it is
targeted to the division site (8). However, our present
observations demonstrate that localization of DivIC is not
essential for its stability under all conditions. Furthermore, our data
suggest that if FtsL stabilizes DivIC (8), then it
does so in the absence of FtsZ.
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FIG. 1.
Western blots showing cellular levels of DivIC
in BB11 (Pspac-ftsZ) (A) and in the divIB null
mutant, SU321, and its wild-type parent strain, SU5 (B). (A) BB11 was
grown at 37°C in IPTG-containing medium (0 min) and resuspended in
either IPTG-lacking or IPTG-containing medium for 20, 40, 60, and 90 min. (B) SU321 and SU5 were shifted from 30°C at mid-exponential
phase to 49°C for 1 h. Samples were collected prior to (0 min)
and 20, 40, and 60 min after the shift. A590
equivalents were loaded in each case. Standard protein positions are
expressed in kilodaltons.
|
|
Localization of DivIB at the division site requires
DivIC.
The dependency of DivIB localization on
DivIC has not been examined previously. DivIC is
essential for growth (16). However, a strain (SU392)
containing a Pspac-divIC gene can readily divide in the
absence of IPTG because wild-type levels of DivIC are not required for division (see below) (15). We have therefore
also examined DivIB localization in a temperature-sensitive
divIC mutant strain, SU391, which contains the
tolR-divIC hybrid gene under Pspac control
(RRC) (15). This hybrid gene encodes a protein comprising the cytoplasmic and membrane domains of the E. coli TolR protein fused to the external C-terminal domain of
DivIC (RRC) (15). At 30 and 45°C
(IPTG-containing medium), wild-type levels of RRC are present in SU391.
However, the strain is temperature sensitive, since division is
inhibited at 45°C in the presence or absence of IPTG. These two
strains thus allowed us to monitor DivIB localization while
altering the levels of mutant or wild-type DivIC. SU391 and
SU392 were grown at 30°C with IPTG. At mid-exponential growth phase,
the cultures were washed and resuspended in media with or without IPTG
and incubated at 30 or 45°C. Samples were taken prior to and 45 min
after resuspension for cell length measurements and IFM. In SU392 cells
(Pspac-divIC) in the presence of IPTG, DivIB
localizations were present in 15% of cells at 30°C and in 21% of
those shifted to 45°C for 45 min (examples of the latter short cells
are shown in Fig. 2). Due to the increase
in frequency of DivIB immunostaining at 45°C, which always
occurred, the relative staining frequencies for all samples were
normalized to that of SU392 grown with IPTG at the same temperature
(Table 1).
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FIG. 2.
DivIB localization in SU391
(Pspac-RRC) and SU392 (Pspac-divIC) in the
presence of IPTG at 45°C. All panels show both SU391 and SU392 cells
(long and short, respectively) that were mixed prior to IFM processing.
(A and C) Phase-contrast images; (B and D) fluorescein
isothiocyanate-immunostained images of the same field. Cells were grown
at 30°C to mid-exponential phase and then shifted to 45°C for 45 min. Arrowheads indicate the long SU391 cells. Bar = 5 µm (A).
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At 30°C in the absence of IPTG, SU392 (Pspac-divIC) cells
contained very little DivIC and were slightly (5%) longer
than those grown in the presence of IPTG (Table 1). Under these
conditions, DivIB localization decreased to 47%. At 30°C
in the presence of IPTG, SU391 cells (Pspac-RRC) were
slightly (15%) longer than SU392 and DivIB localizations
decreased to 77%. This localization decrease was consistently
observed, as was the slight increase in cell length (see also reference
15). In the absence of IPTG at 30°C, SU391 cells
were significantly longer than wild-type cells (three to four times)
and DivIB localizations further decreased to 22%. A
significant proportion (35%) of these localizations occurred where
septa were visible, suggesting that DivIB can assemble only
if enough DivIC is present at the division site to allow septation. Interestingly, we also noticed that in SU391 cells growing
at 30°C in the presence of IPTG, DivIB localizations were extremely faint, much fainter than those in SU392 grown under the same
conditions (not shown). It has been shown previously that at 30°C, in
the presence of IPTG, DivIC midcell staining was
significantly reduced in SU391 compared to SU392, although the cellular
level of the hybrid protein was at least as high as that of the wild
type (15). These observations strongly suggest that at
30°C, DivIB requires DivIC for its localization
and that the efficiency of DivIB localization depends on the
level of DivIC actually assembled at the division site.
At 45°C, SU392 cells (Pspac-divIC) remained short after
the temperature shift in IPTG-containing medium but became slightly filamentous in IPTG-lacking medium at 45°C, averaging 8.12 µm (Table 1). In these longer cells, DivIC is barely detectable by Western analysis. DivIB localization decreased to 35%,
and these localizations were fainter than those observed in the
presence of IPTG at the same temperature. In contrast, SU391
(Pspac-RRC) cells became extremely filamentous when shifted
to 45°C, regardless of whether IPTG was added (22.72 µm in
IPTG-containing medium and 30.40 µm in IPTG-lacking medium),
indicative of a rapid block in cell division under both conditions.
Furthermore, at 45°C in both the presence and absence of IPTG,
essentially no DivIB localized in the long filaments of SU391
(long cells [IPTG-containing medium] in Fig. 2B and D) (Table 1). So
DivIB localization is also dependent on DivIC at
45°C. Furthermore, the level of DivIB localization at
45°C depends on both the cellular level of wild-type DivIC and the ability of DivIC to function. To determine whether
the inability of the RRC protein to support cell division at high temperatures occurs at the level of its assembly at the division site,
it was of interest to examine whether the RRC protein can localize
efficiently in SU391 at 45°C in the presence of IPTG. Although the
RRC protein was not detected at the division site under these
conditions, for unknown reasons even normal levels of wild-type
DivIC are barely detected in strains SU391 and SU392 using
IFM at these high temperatures, making it impossible to draw any such conclusions.
Western blot analysis of cell lysates of SU391 and SU392 revealed that
DivIB levels were unaltered in all samples (data not shown).
This is consistent with the previous finding that DivIB is
stable under conditions in which DivIC is degraded
(8).
Localization of DivIC depends on DivIB only at
high temperatures, but FtsZ localization is independent of
DivIB at all temperatures.
FtsZ and DivIC are
essential for division at all temperatures (4, 16). Since
DivIB is essential only at high temperatures (3),
FtsZ and DivIC must be able to localize to the division site
in the absence of DivIB at low (permissive) temperatures. Can
FtsZ and DivIC localize in the complete absence of
DivIB at the nonpermissive temperature? We examined FtsZ and
DivIC localization in the divIB null strain,
SU321, at 49°C. Although it has been reported previously that
divIB is essential for cell division at temperatures of
>30°C (3), when the originally constructed divIB null mutation from KU608 (3) is present in
our laboratory 168 strain (SU5), it is significantly less temperature
sensitive. In strain SU321, at temperatures of up to 37°C, cell
division is normal and a complete division block is only observed at
49°C (our unpublished observations). The strain-dependent temperature sensitivity of the divIB null mutation has been observed by
others (P. Levin and R. Losick, personal communication).
Exponentially growing cells of SU321 were shifted from 30 to 49°C.
DivIC and FtsZ IFM was performed on cells taken prior to the
temperature shift and at every 20 min after the shift, up to 1 h.
At 30°C, the average cell length of the divIB null strain was 4.42 µm (Table 2), marginally
longer than that of the congenic wild-type strain, SU5, grown under
identical conditions (not shown). Prior to the temperature shift, many
bright DivIC and FtsZ localizations were observed at the
division site (midcell) in both SU5 and SU321, with 37 and 85% of
cells containing DivIC and FtsZ localizations, respectively.
As expected, DivIB is not required for Z-ring formation at
the permissive temperature. It is also concluded that DivIC localization does not require DivIB at the lower temperature
of 30°C, at which DivIB is not essential. These findings
are consistent with the fact that FtsZ and DivIC are
essential for division at all temperatures (4, 16).
At 49°C the wild-type parent strain, SU5, was able to grow and divide
normally, and FtsZ and DivIC localization frequencies never
varied more than twofold. However, division was completely blocked in
SU321 cells at 49°C. Cell length doubled approximately every
generation, and cells were extremely long after 60 min, averaging 45.11 µm (Table 2). The frequency of FtsZ localization did decrease in
these filaments to 52% of the level observed at 30°C (Table 2).
Nonetheless, multiple Z rings formed in the filaments (one filament is
indicated in Fig. 3C and D), with an
average of ~3.8 rings per cell, and these were positioned at
potential division sites (1/2, 1/4, and 1/8, etc., positions)
(data not shown). In contrast, the frequency of DivIC
localizations fell dramatically after the temperature shift, and
by 60 min after the shift, virtually none were observed (Table 2) (long
filament in Fig. 3B).
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FIG. 3.
DivIC and FtsZ localization in SU321
(divIB null) grown at 30°C to mid-exponential phase and
then shifted to 49°C for 1 h. (B and D) Cells immunostained with
fluorescein isothiocyanate; (A and C) phase-contrast image of the same
field. DivIC localization is shown in panel B, while FtsZ
localization is shown in panel D. Cells grown at 30°C (short) were
mixed with long cells (indicated by arrowheads) that had been shifted
to 49°C for 1 h. Bar = 5 µm (A).
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In light of the unexpected finding that extreme temperatures were
required to block septation in SU321, a shift to 45°C was performed
on one of the originally constructed divIB null strains, KU121, that does not grow on plates at 37°C (3). At 45°C
in KU121 cells, a complete block to cell division occurred (data not
shown). The results obtained with respect to both division ability and
localization of DivIC and FtsZ were the same as for SU321
shifted to 49°C (not shown).
Western blot analysis of whole-cell lysates of SU321 (divIB
null) and the wild-type parent strain, SU5, showed that FtsZ levels were very similar in the strains at both temperatures (not shown). Surprisingly however, at 30°C DivIC levels were
consistently two- to threefold higher in SU321 than in SU5 (Fig. 1B).
These levels were further enhanced when SU321 was grown at 49°C for
60 min. Similar results were obtained with KU121 (divIB
null) and its congenic parent strain, tms12+, both prior to
and after a shift to 45°C (not shown).
In conclusion, midcell Z-ring assembly does not depend on
DivIB at 30°C or even at 49°C, when DivIB is
essential for division. DivIC localization to the division
site does not depend on DivIB at 30°C; however, at 49°C,
DivIC is unable to localize in the absence of
DivIB. Thus, DivIC is recruited to the septum site independently of DivIB at 30°C, but at 49°C,
DivIB is required to maintain DivIC at the division
site, perhaps by direct interaction. Increased cellular levels of
DivIC observed in the absence of DivIB raise the
possibility that DivIB negatively influences DivIC synthesis. However, this is not likely to be direct since the only
essential domain of DivIB (80% C-terminal portion) is
external to the cytoplasm (15). Alternatively,
DivIB might somehow decrease the stability of the
DivIC protein.
Our present findings concerning division protein localization in
B. subtilis can be summarized as follows. (i) Z rings can assemble at midcell independently of DivIB, even at higher
temperatures at which DivIB is essential for division. (ii)
Septal recruitment of both DivIB and DivIC requires
FtsZ localization. (iii) DivIB localization requires
DivIC at both 30 and 45°C. (iv) DivIC can be
recruited to the septum site in the absence of DivIB at
30°C but not at 49°C, when DivIB is essential for
division. In other words, DivIB and DivIC are
codependent for localization only at higher temperatures. This suggests
that DivIC is recruited to the septum site earlier than or
together with DivIB at 30°C, but at 49°C, DivIB
is required to maintain DivIC at the division site, perhaps
by direct interaction. A role for DivIB in promoting division complex assembly at higher temperatures has been proposed previously (12, 24). It has been proposed that FtsL and DivIC
interact directly (8). Perhaps all three proteins interact
directly at the septum site. The finding that DivIB
localization efficiency depends on the level of DivIC
assembled at the division site is consistent with the suggestion that
these two proteins interact directly in a fixed stoichiometric ratio at
the site of septum formation. In previous work, the dependency of PBP
2B localization on DivIB and DivIC was examined
only at 48°C (7), so at higher temperatures at least, all
three proteins are interdependent for localization. At lower growth
temperatures, like 30°C, a linear order of localization is still
conceivable, with FtsL localizing first, followed by PBP 2B,
DivIC, and DivIB. Intriguingly, both scenarios are
distinctly different from the division protein assembly pathway in
E. coli (28). What remains to be established
in B. subtilis is whether FtsL localization requires
the other three membrane-bound proteins, presuming FtsL itself is targeted.
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Abstract
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Present address: Research Institute of Molecular Pathology, A-1030,
Vienna, Austria.
