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Journal of Bacteriology, August 2004, p . 5153-5156, Vol . 186,
No . 15
PBP1 Is
a Component of the Bacillus subtilis Cell Division Machinery
Dirk-Jan Scheffers* and Jeffery
Errington
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE,
United Kingdom
Received 29 March 2004/ Accepted 23 April 2004
Bacillus subtilis penicillin-binding protein PBP1 has been implicated
in cell division . We show here that a PBP1 knockout strain is
affected in the formation of the asymmetric sporulation septum and
that green fluorescent protein-PBP1 localizes to the sporulation
septum . Localization of PBP1 to the vegetative septum is dependent on
various cell division proteins . This study proves that PBP1 forms
part of the B . subtilis cell division machinery .
The cell wall is the principal shape-maintaining and stress-bearing
element of the bacterial cell (for reviews, see references 9,
12, and 24) . The Bacillus
subtilis cell wall consists of peptidoglycan (PG), glycan strands
with peptide side chains that are highly cross-linked, and covalently
linked anionic polymers (9) . The polymerization and
cross-linking of PG is mediated by penicillin-binding proteins
(PBPs) . Various studies have indicated that PG synthesis is likely to
be mediated by holoenzyme complexes, comprising PBPs and lytic
transglycosylases that can perform the murein synthesis and
hydrolysis reactions involved in PG synthesis and processing (e.g.,
1, 2, 20,
21, 23) . Since cell wall synthesis during
elongation occurs parallel to the lateral wall, whereas cell wall
synthesis during division occurs perpendicular to the lateral wall,
at least two separate holoenzyme complexes have been proposed (12) .
The putative cell division specific complex of B . subtilis
contains the class B transpeptidase PBP2b (6) and,
together with known cell division proteins, forms the cell division
machinery also known as the "divisome" (14) . In
B . subtilis, the assembly of the cell division machinery starts
with the formation of the FtsZ ring and targeting of FtsA to
this ring (reviewed in reference 7) . Subsequently, all cell
division proteins that contain a transmembrane span, localize
to this ring in a concerted manner . DivIB, DivIC, FtsL, PBP2b, and
probably FtsW (R . A . Daniel and L . J . Wu, unpublished data) are all
completely interdependent for assembly at the division site, and
mutation or depletion of any of these proteins prevents all of the
others from assembling (reviewed in reference 7) .
This is in sharp contrast to Escherichia coli, in which division
proteins assemble in a strictly ordered manner (reviewed in
reference 3) .
Another PBP implicated in cell division in B . subtilis is PBP1,
which localizes to the cell division site (15,
19) . PBP1 is a class A PBP with both
transglycosylase and transpeptidase activities, encoded by the
ponA gene (17) . ponA knockout cells are
not blocked in cell division, but grow more slowly, with an increase
in mean cell length and a decrease in mean cell width (18),
as well as having abnormal septal structures (15) .
These findings indicate that division is suboptimal in ponA
knockout cells . Also, sporulation efficiency is severely reduced in a
ponA knockout compared to single knockouts of genes encoding
other class A PBPs, suggesting that PBP1 may play a role in correct
asymmetric cell division at the start of spore development (18) .
We reasoned that if PBP1 is a true component of the cell division
machinery, it should localize to the asymmetric division site that is
formed during sporulation . Also, given the interdependency of cell
division proteins for assembly at the cell division site in B .
subtilis, we expected PBP1 septal localization to be dependent on
other division proteins .
PBP1 is required for the efficient formation of the asymmetric
sporulation septum. Although PBP1 is not an essential cell division
protein, it may play an important role in the asymmetric cell
division during sporulation . Sporulation efficiency has been reported
to be markedly reduced in a ponA-null mutant (14% compared to
wild type [18]) . To test whether this sporulation
defect is due to the inefficient formation of the asymmetric division
septum, we studied the sporulation properties of a ponA-null
mutant in more detail . Cells were grown in CH medium to an optical
density at 600 nm of
 0.8
before the induction of sporulation by resuspending the cells in
sporulation medium essentially as described previously (22) .
Strain 2083 was grown in the presence of 0.75% xylose and washed with
sporulation medium before resuspension to remove xylose during
sporulation . Although formation of an "axial DNA filament," a marker
of sporulation stage I (16), was not affected in
the ponA-null mutant, the formation of the asymmetric septum
was greatly reduced (Table 1) . A defect in
asymmetric septation should be accompanied by reduced expression of
 F-
and
E-dependent
genes since activation of these sigma factors is dependent on
septation (reviewed in reference 16) . To test
this, the
E-dependent
synthesis of alkaline phosphatase (APase) was measured as described
previously (8, 10) . As shown in
Fig . 1A, the ponA-null mutant showed substantially
delayed and reduced APase activity compared to both the wild-type
strain and the strain expressing green fluorescent protein
(GFP)-PBP1 . Presumably, the defect in asymmetric septum formation is
responsible for the reduced spore frequency observed (Table
1) (18) .
| TABLE 1 . Sporulation properties of a PB1 mutant
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FIG . 1 . (A) Delayed and reduced APase activity in sporulating cells of a
ponA-null mutant . APase activity was measured in strains 168
(wild type,
 ),
2083 (gfp-ponA, •), and 3511 ( ponA,
 ),
with T0 being the moment of resuspension . The results
are representative of three independent experiments . (B) Localization of
GFP-PBP1 to the asymmetric sporulation septum . Samples were obtained 90
min after the induction of sporulation . Images shown include phase
contrast (B), GFP-PBP1 fluorescence (C), DNA staining with DAPI
(4',6'-diamidino-2-phenylindole) (D), and an overlay of the
GFP-fluorescence in green and DNA staining in red (E) . Arrowheads
indicate GFP-PBP1 at asymmetric sporulation septa; asterisks denote
GFP-PBP1 at vegetative division septa . Samples were prepared for
fluorescence microscopy as described previously (19) .
Bar, 5 µm.
|
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Recently, we constructed a fully functional GFP-PBP1 fusion protein
in B . subtilis that localizes to the site of cell division (15,
19) . This GFP-PBP1 strain sporulated with an efficiency
similar to that of the wild-type strain (Table 1) and
displayed a similar APase activity (Fig . 1A),
showing that GFP-PBP1 is fully functional during sporulation as well .
Xylose was not present in the sporulation medium, but GFP-PBP1 is
presumably stable enough to function during sporulation, as
previously described for PBP2b (6) . Previously,
lacZ fusions showed that transcription of ponA decreases
gradually during sporulation to background levels (17),
suggesting that PBP1 does not act later in sporulation .
GFP-PBP1 was visible at asymmetric sporulation septa during the
early stages of sporulation (Fig . 1B to E) . Fluorescence
began to be detected at the asymmetric septum 60 min after the
initiation of sporulation but disappeared after septum closure,
following a localization pattern similar to that previously described
for PBP2b (6) . This makes it likely that, like PBP2b,
PBP1 is targeted to the asymmetric division septum, where it
forms part of the division machinery, and disappears after completion
of the division event .
PBP1 localization depends on other cell division proteins.
Previously, we have shown that GFP-PBP1 septal localization is
dependent on FtsZ (19) . Since FtsZ is essential for the
localization for all other cell division proteins, we tested PBP1
localization in the absence of several membrane proteins that are
components of the divisome, which are all interdependent for
localization . Strains were constructed in which gfp-ponA as
the only functional ponA copy was combined with (i) pbpB
under the control of the Pspac promoter (strain
3542), (ii) a divIB temperature-sensitive (ts) mutation
(strain 3526), and (iii) a divIC ts mutation (strain 3536)
(Table 2) . As shown in Fig . 2B, depletion of
PBP2b led to the disappearance of GFP-PBP1 bands at septa .
Interestingly, occasional spots of GFP-PBP1 could be observed at
incomplete cell division sites (arrow in Fig . 2B),
as previously described for PBP2b (6,
19) . This would indicate that where there is a
limiting amount of PBP2b, sufficient to start septation but not to
support progression of septal ingrowth, PBP1 is also present as part
of the division complex . In the divIB and divIC ts
strains, GFP-PBP1 localization was normal at the permissive
temperature, but upon a shift to the nonpermissive temperature
midcell localization was abolished (Fig . 2D and F) . A control
experiment established that GFP-PBP1 in the wild-type background
is stable at the nonpermissive temperature (results not shown) .
Therefore, we conclude that localization of PBP1 to the division
septum depends on FtsZ, PBP2b, DivIB, and DivIC . FtsL was omitted
from this analysis because the depletion strain for FtsL is dependent
on xylose, as is GFP-PBP1 expression . However, since FtsL localizes
to incomplete septa upon PBP2b depletion and is dependent on DivIB
and DivIC (5), it seems likely that PBP1
localization will also be dependent on FtsL .
| TABLE 2 . Strains used in this study
|
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FIG . 2 . Localization of PBP1 in conditional cell division mutants . (A
and B) pbpB . Strain 3542 was grown in the presence of 0.5 mM IPTG
and 0.75% xylose to exponential phase in S+ medium (19)
at 30°C . The cells were harvested, washed twice, and resuspended in the
same medium with (A) or without (B) IPTG and allowed to grow at 30°C for
90 min to deplete PBP2b . (C and D) divIB . Cells were grown to
exponential phase at 30°C in Difco Antibiotic Medium 3 (17.5 g/liter;
Penassay broth) with 0.75% xylose before the culture was split and grown
for an additional 40 min at a permissive (30°C) (C) or nonpermissive
(48°C) (D) temperature . (E and F) divIC . Cells were grown to
exponential phase at 30°C in Penassay broth with 0.75% xylose before the
culture was split and grown for an additional 40 min at a permissive
(30°C) (E) or nonpermissive (48°C) (F) temperature . Samples were
prepared for fluorescence microscopy as described previously (19) .
Bar, 5 µm.
|
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Finally, PBP1 has both transglycosylase and transpeptidase activities .
We tested whether PBP1 might at least partially substitute for
the essential transpeptidase activity of PBP2b by testing the
sensitivity of a ponA-null mutant to PBP2b depletion compared
to a wild-type strain . ponA-null strains were constructed with
either pbpB under control of Pspac (strain 3907) or
gfp-pbpB under control of Pxyl (strain 3908) .
These strains were grown to exponential phase; the cells were then
harvested, washed, and resuspended in fresh medium with various
concentrations of inducer . Inducer became limiting at similar
concentrations irrespective of the presence of PBP1, with growth
abolished at 0.005 mM IPTG (isopropyl-ß-D-thiogalactopyranoside)
or 0.1% xylose . At slightly higher concentrations of inducer
(0.01 mM IPTG or 0.2% xylose), pbpB expression was enough to
allow wild-type growth rates for the parental strains 3295 and 3122 .
However, in the strains containing both inducible pbpB and the
ponA-null mutation, this level of inducer did not restore the
growth rate to the level of the ponA-null mutation on its own
(a doubling time [Td] of 53 min for strain 3907 at 0.01
mM IPTG compared to a Td of 44 min for strain 3511 [mean
of two experiments]) . This shows that in the absence of PBP1 the
effects of PBP2b depletion are slightly exacerbated .
We show here that PBP1 localization to the vegetative septum is
dependent on various cell division proteins in B . subtilis and
that PBP1 localizes to the asymmetric septum formed during
sporulation . Furthermore, we show that PBP1 plays an important role
in the assembly of the asymmetric division septum during sporulation .
A ponA-null mutant is not inhibited in the entry of
sporulation, as determined by the formation of the axial filament,
but is strongly inhibited in the formation of asymmetric division
septa compared to a wild-type strain . Although other cell division
proteins localize in the absence of PBP1, we conclude that PBP1 forms
a part of the B . subtilis cell division machinery, with a
significant role in the synthesis of septal PG, located perpendicular
to the lateral cell wall . This function is partially redundant during
vegetative growth but becomes more critical during sporulation .
We thank members of our laboratory for discussions and advice .
D.-J.S . is supported by a Marie-Curie Postdoctoral Fellowship
(HPMF-CT-2001-01421) . This study was supported by a grant from the UK
Biotechnology and Biological Sciences Research Council .
* Corresponding author . Mailing address: Molecular
Microbiology, Faculty of Earth and Life Sciences, Vrije Universiteit, De
Boelelaan 1085, 1081 HV Amsterdam, The Netherlands . Phone: 31-20-4446960 . Fax:
31-20-4446979 . E-mail:
dirk-jan.scheffers@falw.vu.nl .
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