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Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382X© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd? 2005593989999Original ArticleSepF, a novel FtsZ-interacting proteinL. W. Hamoen
et al.
Molecular Microbiology (2006) 59(3), 989–999
doi:10.1111/j.1365-2958.2005.04987.x
First published online 30 November 2005
SepF, a novel FtsZ-interacting protein required for a late
step in cell division
Leendert W. Hamoen,1* Jean-Christophe Meile,1,2
Wouter de Jong,1† Philippe Noirot2 and Jeff Errington1
1
Sir William Dunn School of Pathology, University of
Oxford, South Parks Road, Oxford, OX1 3RE, UK.
2
Laboratoire de Génétique Microbienne, INRA, domaine
de Vilvert, 78352 Jouy en Josas Cedex, France.
Summary
Cell division in nearly all bacteria is initiated by polymerization of the conserved tubulin-like protein FtsZ
into a ring-like structure at midcell. This Z-ring functions as a scaffold for a group of conserved proteins
that execute the synthesis of the division septum (the
divisome). Here we describe the identification of a new
cell division protein in Bacillus subtilis. This protein
is conserved in Gram positive bacteria, and because
it has a role in septum development, we termed it
SepF. sepF mutants are viable but have a cell division
defect, in which septa are formed slowly and with a
severely abnormal morphology. Yeast two-hybrid analysis showed that SepF can interact with itself and with
FtsZ. Accordingly, fluorescence microscopy showed
that SepF accumulates at the site of cell division, and
this localization depends on the presence of FtsZ.
Combination of mutations in sepF and ezrA, encoding
another Z-ring interacting protein, had a synthetic
lethal division effect. We conclude that SepF is a new
member of the Gram positive divisome, required for
proper execution of septum synthesis.
Introduction
Cell division in nearly all bacteria is initiated by the polymerization of the conserved tubulin-like protein FtsZ into
a ring-like structure at midcell. This so-called Z-ring functions as a scaffold for the proteins that execute the synthesis of the division septum. The assembly of this
complex and dynamic protein structure, also referred to
as the divisome, has been thoroughly investigated in the
model organisms Escherichia coli and Bacillus subtilis
Accepted 1 November, 2005. *For correspondence. E-mail
[email protected]; Tel. (+44) 1865 275 562; Fax
(+44) 1865 275 556. †Present address: Groningen Biomolecular
Sciences and Biotechnology Institute, University of Groningen,
Kerklaan 30, 9751 NN Haren, the Netherlands.
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd
(reviewed in Errington et al., 2003). Most of the components of the divisome are conserved; however, there are
distinct differences between the mechanisms of divisome
assembly in B. subtilis and E. coli. It is likely that this
reflects structural differences between the cell-envelopes
of Gram positive and Gram negative bacteria. We will
focus here on cell division in B. subtilis.
The divisome complex spans the cytoplasmic membrane. At the core of the cytoplasmic site of the divisome
lies the Z-ring, assembly of which is supported by at least
two other cytosolic proteins. Most important is the conserved actin-like division protein FtsA. This protein is
essential and interacts with the C-terminus of FtsZ (Erickson, 2001). It has recently been shown that the conserved
C-terminus of FtsA contains an amphipathic helix that is
essential for targeting of the protein to the membrane. This
led to the suggestion that FtsA serves as the principal
membrane anchor for the Z-ring (Pichoff and Lutkenhaus,
2005). In addition, there is a small 85-amino-acid cytoplasmic protein, ZapA, which promotes polymerization of
FtsZ. However, a deletion of zapA showed no apparent
phenotype in B. subtilis or E. coli (Gueiros-Filho and
Losick, 2002; Johnson et al., 2004). EzrA is another conserved protein that binds to the Z-ring (Levin et al., 1999).
This large protein has a single N-terminal transmembrane
domain, but this domain is not required for interaction with
FtsZ (Haeusser et al., 2004). The role of EzrA in the
activity of the divisome is not entirely clear. An ezrA
mutant is viable, yet it tends to assemble multiple Z-rings
both at polar and midcell positions. It is assumed that this
protein is a negative regulator of Z-ring formation (Levin
et al., 1999).
At least two of the conserved B. subtilis divisome proteins are involved in peptidoglycan synthesis: the penicillin-binding protein Pbp2B and the integral membrane
protein FtsW (R.A. Daniel and J. Errington, unpublished;
Daniel et al., 2000; Aarsman et al., 2005). The 10 membrane spanning domains of FtsW suggest a possible role
in transport, and it has been speculated that FtsW translocates the lipid-linked precursor for the septal peptidoglycan matrix (Holtje, 1998). The periplasmic part of the
divisome comprises, aside from Pbp2B, three other conserved cell division proteins: FtsL, DivIB and DivIC. Like
Pbp2B, these proteins are attached to the membrane by
a single transmembrane domain. Not much is known
about the function of these three proteins. They seem to
990 L. W. Hamoen et al.
be intrinsically unstable and biochemical data suggest
that they form heteromultimers that ensure their stability
(Sievers and Errington, 2000; Robson et al., 2002). Therefore, it has been proposed that these proteins may fulfil a
regulatory role in divisome assembly and/or disassembly.
The divisomes of B. subtilis and E. coli appear to be
comparable in composition (Errington et al., 2003). However, the E. coli divisome comprises several more components, such as FtsK, FtsE, FtsQ, FtsN and AmiC
(Aarsman et al., 2005). This complexity could be a consequence of the fact that in E. coli cytokinesis requires constriction of outer membrane, as well as inner membrane,
and peptidoglycan layers. An intriguing difference
between B. subtilis and E. coli is the way their divisomes
assemble. In E. coli, the recruitment of the different components seems to occur in an ordered manner, whereby
the depletion of one cell division protein leads to the
absence of the downstream proteins in the interaction
pathway [the order being: FtsZ (FtsA, ZipA, ZapA), FtsK,
FtsQ (FtsL, FtsB), FtsW, FtsI, FtsN and AmiC] (Aarsman
et al., 2005; Goehring et al., 2005). This linearity of
recruitment is not observed in B. subtilis. The assembly of
the B. subtilis divisome seems to depend on the presence
of all the essential division proteins, and depletion of FtsA,
DivIC, FtsL or Pbp2B, abolishes the positioning of the
other cell division proteins at midcell. Only the polymerization of the Z-ring does not require the other division
proteins (Errington et al., 2003). Although much is known
about the assembly of the divisome, the actual mechanism of constriction remains unclear. The homology of
FtsZ and FtsA with tubulin and actin, respectively, makes
it plausible that these proteins lead the constriction of the
divisome. However, whether they are sufficient to drive
this process, or whether the synthesis of the peptidoglycan provides the ultimate force for constriction, remains
to be established (Bramhill, 1997; Errington et al., 2003).
In this paper we describe the identification of a new
FtsZ-interacting cell division protein, which we designated
SepF. Combination of a sepF and ezrA mutation results
in a synthetic-lethal division defect, suggesting that one
or the other of these proteins is necessary for proper
functioning of the Z-ring. However, sepF single mutants
affect only the later stages of septum constriction indicating that SepF is important throughout the process of division. While this work was in progress, we learned that
Ogasawara and coworkers had obtained similar results
implicating SepF in cell division of B. subtilis.
Results
Deletion of the ylmB-H region has a mild cell division
defect
In rod-shaped bacteria the conserved protein couple
MinC/MinD prevents aberrant Z-ring assembly close to the
poles of the cell. Mutations in these proteins result in polar
division and anucleate minicells. MinC is the actual inhibitor of FtsZ assembly but requires the membrane-bound
ATPase MinD for its activity (Hu et al., 1999; Marston and
Errington, 1999). In B. subtilis the activity of this protein
couple is confined to the cell poles due to interactions with
the polar localized protein DivIVA (Edwards and Errington,
1997). How DivIVA accumulates at the poles is unknown.
In many Gram positive bacteria the conserved ylm genes
are found upstream of divIVA (Fig. S1). Several of the ylm
genes have been disrupted previously (Kobayashi et al.,
2003) (see also, http://genome.jouy.inra.fr/cgi-bin/micado/
index.cgi). However, none of the mutants were reported
to have a phenotype. The genes ylmE and ylmH were not
tested, and to assess a potential role of the ylm genes in
DivIVA localization, we replaced the complete ylmB-H
region with an antibiotic resistance marker (hereafter
referred to as ylm mutant, Fig. 1). Light microscopic
observations of the resulting strain (B. subtilis 3317) seem
to indicate a normal wild-type phenotype, although the
cells appeared longer. To analyse this in more detail, we
measured cell length of an ylm mutant and wild-type culture. As shown in Fig. 1C and D, the difference was modest during exponential growth, with an average length of
mutant cells approximately 20% longer than wild-type
cells (4.6 and 5.5 µm for wild-type and ylm mutant respectively). In the stationary phase this difference increased to
about 40% (2.8 and 4.0 µm for wild-type and ylm mutant
respectively). It was clear that the ylm genes were not
required for DivIVA localization because a normal polar
and septal fluorescent pattern was observed when the ylm
deletion was introduced into a strain carrying a divIVA-gfp
fusion (Fig. 1F). However, there was a clear difference in
spacing of green fluorescent protein (GFP) bands, which
reflects the increase in cell length of the mutant.
Disruption of the ylm locus has a synthetic lethal effect
when combined with ezrA
It seemed possible that the mild mutant phenotype produced by the ylm deletion might be exacerbated when
combined with other mutations that impair cell division. To
test whether the ylm mutant had increased sensitivity to
FtsZ levels, the ylm deletion was introduced into cells in
which ftsZ expression is under control of the IPTG-inducible Pspac promoter. Although low IPTG concentrations
resulted in elongated cells, the presence of an ylm deletion did not substantially aggravated this phenotype. Various other mutations in genes that regulate FtsZ
polymerization, such as minC and zapA, were combined
with an ylm deletion, but the effects on cell length were
again marginal (data not shown). In contrast, the number
of transformants was much lower than expected when the
ylm mutant was transformed with chromosomal DNA from
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
SepF, a novel FtsZ-interacting protein 991
2 kb
A
ylmA
-G
-B -C -D -E -F -H
sigE sigG
divIVA
B
ylmA
ileS
kmR
ylm mutant
sigE sigG
divIVA
Fig. 1. Phenotype of an ylm mutant. The organization of the ylm
locus and adjacent genes is shown in the upper panel (A). In the ylm
mutant (strain 3317) the ylmB-H region has been replaced by a
kanamycin-resistance marker (kmR) (B). The positions of putative
terminator sequences are indicated. Graphs C and D show cell length
measurements of a wild-type culture and an ylm mutant culture.
Samples were taken during logarithmic growth (C), and after 2 h in
stationary phase (D). The numbers of cells measured (n) are indicated, and cell lengths are given in µm. The fluorescence microscopy
pictures show DivIVA-GFP expression in wild-type (E) and ylm mutant
cells (F). Scale bar indicates 5 µm.
ileS
Deletion of the ylm locus leads to impaired septa
25
ylm (n = 273)
C
frequency (%)
20
wild type (n = 260)
15
10
5
<
1-1 1
1.5 .5
2.0 2.0
-2.
2.5 5
3.0 3.0
-3.
5
3.5
4-4 4
.
4.5 5
-5
5-5
.5
5.5
6-6 6
.5
6.5
-7
7-7
.
7.5 5
-8
8-8
.
8.5 5
-9
9-9
.
9.5 5
>
0
cell length
35
ylm (n = 265)
frequency (%)
30
wild type (n = 282)
D
25
20
In view of the possible deficiency in cell division resulting
from deletion of the ylm locus, the ultrastructure of division
septa was analysed using electron microscopy (EM). It
appeared that in ylm mutant cells septa showed strong
deformations (Fig. 2). This was most apparent in the early
stages of septum synthesis, as shown in Fig. 2D and E.
Division seemed to proceed normally when septation has
been completed, although distortions in matured septa
were still observed (Fig. 2F). These distortions did not
seem to lead to deformed cell poles (data not shown).
The ylmD-H genes comprise an operon
Inspection of the DNA sequence of the conserved ylm
region revealed only a single large space likely to accom-
15
wt
10
5
<1
1-1
1.5 .5
-2
2.0 .0
-2.
2.5 5
-3
3.0 .0
-3.
5
3.5
-4
4-4
.5
4.5
-5
5-5
.5
5.5
-6
6-6
.5
6.5
-7
7-7
.5
7.5
-8
8-8
.5
8.5
-9
9-9
.5
9.5
>
0
cell length
E
wt
F
Dylm
A
B
C
E
F
H
I
D
D
ylmF
G
an ezrA mutant. The few colonies that emerged appeared
to have lost the kanamycin-resistance marker, and therefore had been co-transformed with the wild-type ylm locus
from the donor strain. This finding supported the view that
one or more of the ylm genes plays a role in cell division.
Fig. 2. Transmission electron microscopy (TEM) images of division
septa in wild-type cells (A–C), in the ylm mutant (B. subtilis 3317) (D–
F) and in the ylmF:pMUTIN4 integration mutant (B. subtilis BFA2863)
grown in the presence of 1 mM IPTG (G–I).
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
992 L. W. Hamoen et al.
ylmD
wt
mut
ylmG
wt
mut
ylmH
wt
mut
divIVA
wt
mut
Fig. 3. Northern analysis of the ylmD-divIVA region. Total RNA from
wild-type (wt), and a B. subtilis mutant (mut) that contains the Pspac
promoter immediately upstream of the ylmD open reading frame
(B. subtilis 4055), was hybridized with radioactively labelled probes
directed against ylmD, ylmG, ylmH and divIVA respectively. B. subtilis
4055 was grown in the absence of IPTG.
modate a promoter: a 165 bp non-coding region between
ylmC and ylmD. To test whether this region contains a
promoter, and simultaneously, to examine whether ylmB
and/or ylmC contributed to the cell division defect, two
deletion mutants were constructed: strains 4054 and
4055. In strain 4055, ylmB, and ylmC including the 165 bp
non-coding region, were replaced with the IPTG-inducible
Pspac promoter. Strain 4054 was similar except that the
165 bp potential promoter region remained in position
upstream of ylmD. When an ezrA mutation was introduced
into these strains, the resulting transformants grew normally in the presence of IPTG. However, transformants
from strain 4055 became elongated and eventually lysed
when IPTG was omitted, whereas those of 4054 retained
normal cell length and viability (data not shown). These
results showed that loss of ylmB and ylmC does not give
a division phenotype, and strongly suggested that the
gene or genes responsible for the division effect lay downstream of the 165 bp region and required transcription
from that promoter (ylmD promoter).
To test which of the genes downstream of the 165 bp
region were transcribed from the putative promoter, RNA
was extracted from wild-type B. subtilis, and strain 4055
(grown in the absence of IPTG). As shown in Fig. 3,
probes directed against ylmD, ylmG and ylmH gave a
clear signal when RNA from wild-type cells was used, but
almost no signal when RNA from the mutant was used.
This suggests that there is a single polycistronic mRNA,
and that the promoter for this mRNA is absent from strain
4055. In contrast, a probe against the downstreamlocated gene divIVA hybridized only to a faster migrating
band that was present in both strains. The latter supported
previous findings that divIVA has its own promoter
(Edwards and Errington, 1997).
ylmF (sepF) is responsible for the division defect
To determine which of the ylm genes was responsible for
the effects on cell division, single gene disruptions were
constructed. To reduce polar effects, the integration vector
pMUTIN4 (Vagner et al., 1998), which places downstream-located genes under control of the Pspac promoter, was inserted by single (Campbell type) crossover.
ylmG proved to be too small for efficient homologous
recombination and was deleted by double crossover.
Chromosomal DNA from the various ylm mutants (listed
in Table 1) was used to transform an ezrA mutant, and
transformants were selected on plates containing 1 mM
IPTG. Normal transformation percentages were found
with DNA from all ylm integration mutants except for the
ylmF integration. Construction of an ylmF ezrA double
mutant proved not to be possible. Electron microscopic
inspection of the ylmF:pMUTIN4 integration mutant
(Fig. 2G–I) indicated that deletion of this gene was
responsible for the aberrant structure of septa observed
in the ylm mutant. As it appeared that ylmF is involved in
septum synthesis hereafter we refer to it as ‘sepF ’.
SepF interacts with itself and FtsZ
In most Gram positive bacteria sepF lies in a conserved
gene cluster together with ylmE, ylmG and ylmH homologues (see Fig. S1). This suggested that their gene products might be functionally related. To assess whether
SepF, YlmE, -G and -H physically interacted, the corresponding open reading frames were expressed in yeast
as fusions to the GAL4 activation domain (AD), and to the
GAL4 binding domain (BD). Interactions were tested by a
yeast two-hybrid mating assay, as described in Experimental procedures. The result of a typical mating experiment is shown in Fig. 4A. The self-activation of the
interaction reporter genes by the BD-YlmH fusion prevented any interpretation for this protein. In contrast, BDSepF interacted specifically with AD-SepF, suggesting
that the protein can dimerize or oligomerize. Various division proteins were then tested for interaction with SepF,
YlmE and YlmG, including: DivIB, DivIC, DivIVA, EzrA,
FtsA, FtsL, FtsZ, Pbp2B and ZapA. Only a single interaction emerged from this extensive screening experiment: a
clear interaction between SepF and FtsZ (Fig. 4B). This
result provided strong support for the notion that SepF is
part of the divisome complex.
SepF localizes to division sites dependent on FtsZ but not
later components of the divisome
To further test the interaction of SepF with FtsZ, we examined the localization of the protein in vivo by fusing it to
GFP. As shown in Fig. 5A, cells expressing a sepF-gfp
fusion showed a pattern of regular transverse bands
(arrowheads) and dots (arrows) corresponding to septa in
the early and late stages of division. Unfortunately, the
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
SepF, a novel FtsZ-interacting protein 993
Table 1. Bacillus subtilis strains used in this study.
B. subtilis
Relevant genotypea,b
Construction or reference
3317
1803
3318
1801
3362
4054
4055
4076
4077
BFA2813
4214
BFA2863
4094
4215
4181
4216
BFA2817
4217
2020
4218
3122
4219
ylmB-H::km
divIVA::(PdivIVA-gfp divIVA+ cat)
ylmB-H::km divIVA::(PdivIVA-gfp divIVA+ cat)
chr::(Pspac-ftsZ ble)
ezrA::tet
ylmBC::(pMut erm)
ylmBC::(pMut Pspac-ylmD erm)
ylmBC::erm ezrA::tet
ylmBC::(erm Pspac-ylmD) ezrA::tet
ylmD:(pMUTIN Pspac-ylmE erm)
ylmE:(pMut Pspac-sepF erm)
sepF(ylmF):(pMUTIN Pspac-ylmG erm)
ylmG::(pMut Pspac-ylmH erm)
ylmH:(pMut erm)
amy::(Pxyl-sepF-gfp spc)
amy::(Pxyl-sepF-gfp spc) chr::(Pspac-ftsZ ble)
yllB:(pMUTIN Pspac-ylxA-ftsL-pbp2B erm)
amy::(Pxyl-sepF-gfp spc) yllB:(pMUTIN Pspac-ylxA-ftsL-pbp2B erm)
amyE::(Pxyl-gfp-ftsZ spc)
ylmBC::(erm Pspac-ylmD) ezrA::tet amyE::(Pxyl-gfp-ftsZ spc)
pbp2B::(Pxyl-gfp-pbp2B cat)
ylmBC::(erm Pspac-ylmD) ezrA::tet pbp2B::(Pxyl-gfp-pbp2B cat)
This study
Thomaides et al. (2001)
3317 transformed with 1803
Marston et al. (1998)
This study
This study
This study
4054 transformed with 3362
4055 transformed with 3362
BFA project
This study
BFA project
This study
This study
This study
4181 transformed with 1801
BFA project
4181 transformed with BFA2817
J. Sievers (unpublished)
4077 transformed with 2020
Scheffers et al. (2004)
4077 transformed with 3122
a. All strains are based on 168 and carry the trpC2 marker.
b. Antibiotics used: km, kanamycin; cat, chloramphenicol; ble, phleomycin; erm, erythromycin; spc, spectinomycin; and tet, tetracycline.
SepF-GFP fusion did not complement a sepF disruption
in an ezrA background. Nevertheless, because the fusion
localized at division sites, independent of the presence of
wild-type SepF (data not shown), we assume that the
localization pattern, at least partially, reflects the normal
localization of the protein. To test whether the localization
was dependent on FtsZ, the GFP fusion construct was
moved into a strain in which ftsZ could be repressed.
Depletion of FtsZ resulted in the formation of long filaments devoid of septal SepF-GFP (Fig. 5C).
To examine whether SepF localization also required a
later step in divisome assembly, the sepF-gfp construct
was introduced into a strain in which the division proteins
FtsL and Pbp2B could be simultaneously depleted. This
leads to a rapid block in assembly of the divisome, but Zrings remain present at intervals in the resulting elongated
cell filaments (Daniel et al., 1998; 2000). The membrane
staining in Fig. 5F shows that depletion of FtsL and Pbp2B
results in elongated cells devoid of septa. However, the
GFP signal in Fig. 5E indicates that in these cells SepF
bands remained visible. Thus, SepF does not require the
later assembling components of the divisome to be
recruited to the Z-ring.
SepF acts late in the division process
To determine at what step SepF intervenes in division, we
first tested whether Z-rings are still formed in the absence
of SepF (and EzrA). As shown in Fig. 6B, cells became
elongated and no longer divided when the expression of
SepF was repressed in the absence of EzrA. However, Z-
rings still assembled, as visualized with an ftsZ-gfp fusion
(Fig. 6A). Some of the Z-rings appeared abnormal, like
double structures or short helices (arrowheads). Such
abnormal structures were also observed in ezrA single
mutants, but they appeared more often when SepF was
depleted, as well. Nevertheless, the conclusion must be
that SepF is not essential for the formation of Z-rings in
an ezrA mutant. To test whether SepF affects a later step
in division, the experiment was repeated with a gfp-pbp2B
fusion. As shown in Fig. 6E and F, bands of GFP-Pbp2B
(arrows) were still visible after depletion of SepF, albeit
with a reduced intensity. The persistence of the GFPPbp2B banding pattern suggests that SepF is not required
for assembly of the divisome under these conditions.
The GFP experiments suggested that the sepF ezrA
arrest in division occurs at a very late step. EM was used
to examine the ultrastructure of the arrested cells. In the
presence of IPTG division septa had a fairly normal
appearance, indicating that deletion of ezrA does not significantly affect the formation of the division septum
(Fig. 6I–K). In the absence of IPTG, normal division septa
were essentially absent. In some cells the early stages of
constriction were visible (Fig. 6L–N). It is likely that these
are the remains of septa that had been initiated just before
SepF became limiting.
Discussion
The ylm locus in B. subtilis and related bacteria
In many bacteria the genes of the main players in cell
division and cell wall synthesis are located together in a
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
994 L. W. Hamoen et al.
A
YlmE
SepF
YlmG YlmH ad
YlmE
SepF
YlmG
YlmH
bd
B
FtsZ
FtsA
EzrA
ZapA
DivIVA
ad
YlmE
SepF
YlmG
Most Gram negative bacteria contain homologues of
ylmE and ylmG, but they lack genes that show similarities
to sepF or ylmH. Interestingly, cyanobacteria harbour cell
division proteins that are typical of both Gram positive and
Gram negative bacteria. In the genomes of several
sequenced cyanobacteria, homologues of both divIVA
and minE are present, and sepF, ylmE, ylmG and ylmH
are all conserved (Miyagishima et al., 2005). However,
these genes are not clustered at a single locus in the
genome. Despite their outer membrane, molecular phylogenetic studies now group the cyanobacteria separately
from the other Gram negatives (Woese et al., 1990; Miyagishima et al., 2005). It was recently shown that in the
cyanobacterium Synechococcus elongatus, an interruption of sepF resulted in a doubling in cell length (Miyagishima et al., 2005). The relatively mild effect of sepF
disruption on cell length in B. subtilis may explain why this
gene had not been picked up in previous mutant searches.
Deletion of sepF resulted in aberrant septum synthesis
according to the EM images of Fig. 2. These structural
deformations could delay maturation of the division septum, which may explain why sepF mutations result in
longer cells.
A late role for SepF in cell division
bd
Fig. 4. Yeast two-hybrid interactions. Diploid yeast cells expressing
the indicated combinations of proteins fused to the GAL4 BD, and to
the GAL4 AD, were subjected to selection for expression of the ADE2
interaction reporter. Binary interactions appeared as pairs of growing
colonies on selective SC-LUA medium (Synthetic Complete medium,
lacking leucine, uracil and adenine). The upper matrix (A) presents
an example of an interaction study between SepF and the conserved
Ylm proteins. The lower matrix (B) shows a screen in which possible
interactions with other division proteins are tested. On top of the
matrix the activator fusions are indicated, and at the left site the BD
fusions are indicated. Every BD/AD combination was tested in duplicate. Control matings with the GAL4 BD (bd) and GAL4 AD (ad) were
performed to detect self-activation.
cluster that is well conserved, the DCW cluster (Massidda
et al., 1998). In many Gram positive bacteria the ylm
locus is located next to the DCW cluster (Fig. S1). An
indication that the ylm genes might have a role in cell
division came from a deletion study in Streptococcus
pneumoniae (Fadda et al. 2003). In this organism ftsA,
ftsZ, the ylm genes, and divIVA form an operon. Deletion
of divIVA resulted in a strong morphological effect with
incomplete septa, strongly deformed cells and occasional
anucleate cells. Insertions in the individual ylm genes
resulted in thin septa, and a slightly altered cell shape.
Unfortunately, interpretation of these data was difficult
due to potential polar effects on the expression of ylm
genes and divIVA.
In this study we have shown that SepF is an important
cell division protein. The conserved genomic localization
of sepF upstream of divIVA might suggest a functional
relationship. However, deleting sepF does not result in a
minicell phenotype, and the typical targeting of DivIVA to
division sites and cell poles was unaffected in such
mutant. In addition, deletions of divIVA and minC/D did
not interfere with the localization of SepF (data not
shown). This suggests that SepF functions in a different
pathway from DivIVA. The information that can be
extracted from the primary sequence of the ylm operon is
rather limited. Only ylmE shows sequence homology that
could point towards a specific activity in the cell division
process, because this protein has homology with pyridoxal phosphate-dependent enzymes. The cofactor pyridoxal phosphate is involved in a wide variety of catalytic
reactions, including the conversion of L-alanine to Dalanine by alanine racemases, which is one of the first
steps in the synthesis of peptidoglycan (Shaw et al.,
1997). Nevertheless, the yeast two-hybrid study did not
reveal an interaction between SepF and YlmE, YlmG or
YlmH. In addition, none of the ylmE, ylmG or ylmH
mutants showed a filamentous phenotype in an ezrA
background that would resemble a sepF ezrA double
knockout. So far, our data do not provide support for the
assumption that SepF and the other Ylm proteins function
in the same pathway, as the conserved operon organization would suggest.
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
SepF, a novel FtsZ-interacting protein 995
A
wt
Fig. 5. In vivo localization of SepF. Upper panel
(A) shows SepF-GFP expressed in wild-type
cells (B. subtilis 4181). The arrowheads and
arrows indicate septa in early and late stages
of division respectively. Membranes were
stained with the fluorescent membrane dye
FM5-95 (B). The fluorescence images C (SepFGFP) and D (membranes) show cells that were
depleted for FtsZ (B. subtilis 4216). The fluorescence images E (SepF-GFP) and F (membranes) show cells that were depleted for FtsL
and Pbp2B (B. subtilis 4217). Depletion was
achieved by growing cells for 2 h in the absence
of IPTG at 30°C. Scale bars indicate 5 µm.
B
C
E
- ftsZ
- ftsL/pbp2B
D
F
To facilitate the discussion on the function of SepF, we
dissect the process of septum formation into three
sequential steps: (i) Z-ring formation, (ii) divisome assembly and (iii) septum synthesis (Fig. 7). The first step, the
formation of the Z-ring, begins with the polymerization of
FtsZ monomers into protofilaments that subsequently
assemble into a Z-ring. FRAP studies (fluorescence
recovery after photobleaching) have shown that the Z-ring
is a dynamic structure with a turnover of subunits within
seconds (Stricker et al., 2002; Anderson et al., 2004). This
remodelling of the Z-ring appears to depend on the hydrolysis of GTP by FtsZ polymers (Stricker et al., 2002; Anderson et al., 2004). Several proteins control the assembly of
the Z-ring in B. subtilis such as MinC and ZapA (Fig. 7).
In vitro experiments with MinC of E. coli have shown that
this protein interacts with FtsZ and inhibits polymerization
(Hu et al., 1999). EzrA is also a negative regulator of Zring formation, and recent biochemical experiments
showed that purified EzrA inhibits the polymerization of
FtsZ into protofilaments (Haeusser et al., 2004). ZapA has
a positive effect on Z-ring formation and in vitro studies
showed that this protein strongly stimulates the bundling
of protofilaments (Gueiros-Filho and Losick, 2002). Interestingly, an ezrA zapA double knockout is highly filamen-
tous (Gueiros-Filho and Losick, 2002). The reason for this
is not clear, but this phenotype could be used to argue
that SepF might have a comparable function with that of
ZapA. However, we do not think that this is the case. A
zapA mutant is sensitive to reduced FtsZ concentrations,
and this was not observed for the ylm mutant. Also the
introduction of minC or zapA deletions into the ylm mutant
did not affect cell length more than the mutations did
individually. Finally, in the absence of both SepF and EzrA,
Z-rings are still formed. Therefore, we assume that SepF
is not involved in Z-ring formation, but is active in a later
step of septum synthesis. Interestingly, SepF requires
only the Z-ring for its localization, which is so far unique
for a late cell division protein in B. subtilis.
After the Z-ring has formed the divisome assembles. In
E. coli there is a considerable time delay between these
two steps: 15–20 min, depending on the growth rate
(Aarsman et al., 2005). Whether this is also the case for
B. subtilis is not known. As described in Introduction,
assembly of the divisome in this organism is a cooperative
process and requires the presence of a number of essential division proteins (Fig. 7). SepF is not essential for this
step of the process, because the ylm mutant grew normally. Even when both SepF and EzrA are absent, and
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
996 L. W. Hamoen et al.
A
B
+ IPTG
C
- IPTG
D
E
F
- IPTG
G
+ IPTG
H
+ IPTG
I
J
K
- IPTG
septation is blocked, the divisome still assembles, as the
fluorescent GFP-Pbp2B bands in Fig. 6E indicated. Thus,
it is unlikely that the main function of SepF is to stimulate
the assembly of the divisome complex. However, the EM
images of Fig. 2 suggest that the synthesis of the division
septum occurs in an irregular manner when SepF is
absent. Such aberrant septa have not been described for
B. subtilis cell division mutants. Although mutants that
affect Z-ring formation can result in misplaced septa, they
do not show such strong deformations and thickening of
division septa. For example, Fig. 6I–K illustrates that
septa have a wild-type appearance in an ezrA background, whereas it is clear from Fig. 2 that maturation is
strongly affected in a sepF mutant septum. Therefore, we
favour a model in which SepF participates in the third step
of the cell division process: the constriction of the divisome and/or synthesis of the septum wall.
The reasoning behind the model of Fig. 7 presents us
with a problem, namely, how to explain the synergistic
effect of an ezrA sepF double knockout. The precise role
that EzrA fulfils in the division process is not entirely clear.
Fluorescence microscopy studies have shown that EzrA
primarily localizes at the site of cell division, and that this
localization is FtsZ-dependent (Levin et al., 1999). This
suggests that EzrA is active during synthesis of the septum, which is surprising because EzrA is an inhibitor of
FtsZ polymerization. However, biochemical experiments
provided an explanation for this paradox because EzrA is
unable to disassemble preformed FtsZ-polymers in vitro
(Haeusser et al., 2004). The polymerization of FtsZ is very
sensitive to differences in cellular FtsZ concentrations,
and even a twofold increase can result in doublet and
polar Z-rings (Weart and Levin, 2003). A local increase in
the FtsZ concentration in the vicinity of a dynamic and
contracting Z-ring could therefore lead to the assembly of
a doublet, which could eventually result in the formation
of a minicell. Possibly, EzrA represses formation of adja-
Z-ring
L
M
Divisome
N
Fig. 6. Depletion of SepF in an ezrA background. The effect on Zring assembly is presented In the upper panel. B. subtilis strain 4218
(ezrA, Pspac-ylmD-H, Pxyl-gfp-ftsZ) was grown in the absence (A, B)
or presence of 1 mM IPTG (C, D), for 2 h at 30°C. Arrowheads
indicate aberrant Z-rings. The fluorescence images in the middle
panel were taken from cells expressing a GFP-Pbp2B fusion
(B. subtilis 4219: ezrA, Pspac-ylmD-H, Pxyl-gfp-pbp2B). The strain
was grown in the absence (E, F) or presence of 1 mM IPTG (G, H),
for 2 h at 30°C. Some of the GFP-Pbp2B bands are pointed out by
arrows. Membranes were stained with fluorescent membrane dye
FM5-95 (B, D, F, H). Scale bars indicate 5 µm. The TEM images in
the lower panel show some division septa of B. subtilis 4077 (ezrA,
Pspac-ylmD-H) grown in the presence of 1 mM IPTG (I J, K) or
absence of IPTG (L, M, N).
MinC
( EzrA )
ZapA
FtsZ
FtsA
FtsL
DivIB
DivIC
FtsW
Pbp2B
SepF
Fig. 7. A model of the different stages leading to septum synthesis.
The process is divided in three subsequent steps: Z-ring formation,
divisome assembly, and septum synthesis accompanied by constriction of the Z-ring. Grey and black lines indicate cell wall and cytoplasmic membrane respectively. Open circles indicate FtsZ molecules,
and the different proteins that constitute the divisome are depicted as
open rectangles.
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
SepF, a novel FtsZ-interacting protein 997
cent Z-rings when septum synthesis is in progress. Thus,
EzrA could be most important during the period when
SepF is also active. Blocking both processes may therefore be too much to bear for the division machinery.
In conclusion, SepF is a new member of the divisome
of Gram positive bacteria. We postulate that it acts as a
late division protein, even though it is an early recruit to
the Z-ring. We now need to know whether the protein is
required for divisome constriction, or plays a more direct
role, perhaps in septal peptidoglycan synthesis.
Experimental procedures
General methods and materials
Molecular cloning, polymerase chain reactions (PCR) and
E. coli transformations were carried out using standard techniques. B. subtilis strains were grown in Difco antibiotic
medium 3 (PAB). E. coli was used as cloning intermediate.
B. subtilis chromosomal DNA for transformation and PCR
reactions was purified as described by Venema et al. (1965).
Transformation of competent B. subtilis cells was performed
using an optimized two-step starvation procedure based on
the method of Anagnostopoulos and Spizizen (Anagnostopoulos and Spizizen, 1961; Hamoen et al., 2002). Selection
for E. coli and B. subtilis transformants was carried out on
nutrient agar supplemented with: 100 µg ml−1 ampicillin,
5 µg ml−1 kanamycin, 4 µg ml−1 erythromycin, 5 µg ml−1
chloramphenicol, 50 µg ml−1 spectinomycin, 0.2 µg ml−1 phleomycin or 5 µg ml−1 tetracyclin.
ylmC1 and ylmF2. The amplified products were digested,
ligated and transformed to B. subtilis 168. In case of
B. subtilis 4054 only the ylmB-C region was replaced by
double crossover by pMut. For this, the upstream region was
amplified by primers sigE1 and ylmA2, and the downstream
region was amplified by primers ylmD3 and ylmF2. Insertion
deletion of ylmE (B. subtilis 4214) and ylmH (B. subtilis 4215)
were obtained by single crossover of pMut. An internal fragment of ylmE was amplified using primers ylmE8 and ylmE9.
pMut was again amplified from pMUTIN4 using primers
pmut1 and pmut2. After digestion and ligation the ligation
mixture was first transformed to E. coli, and plasmid DNA
from the correct clone was transformed to B. subtilis 168. The
same procedure was followed for the construction of the ylmH
integration except that pMut was amplified using primers
pmut4 and pmut5, and an internal ylmH fragment was
obtained using primers ylmH11 and ylmH14. In case of the
ylmG deletion (B. subtilis 4094), a double crossover was
required. The upstream region was amplified by primers
ylmD1 and ylmF8, and the downstream region was amplified
by primers ylmH15 and ileS10. pMut was again amplified
from pMUTIN4 using primers pmut1 and pmut2. The amplified products were digested, ligated and transformed directly
to B. subtilis 168. All chromosomal integrations were verified
by PCR, restriction digestion, and sequencing. For the construction of a sepF-gfp fusion (B. subtilis 4181), we made use
of the B. subtilis GFP-cloning vector pSG1154 (Lewis and
Marston, 1999). The sepF open reading frame was amplified
using primers ylmF1 and ylmF4. Cloning this fragment into
pSG1154 resulted in a C-terminal GFP fusion under control
of the xylose-inducible Pxyl promoter. After transformation to
B. subtilis 168, the gene fusion was integrated at the amy
locus by double crossover.
Strain construction
The relevant B. subtilis strains are listed in Table 1. Primers
used for PCR reactions are listed in Table S1. Deletion of the
ylmB-H region (B. subtilis 3317) was accomplished by double
crossover of a kanamycin marker. The upstream region was
amplified by PCR using primers IIGA and ylmB2, the downstream region was amplified using primers ylmH1 and ileS2,
and the kanamycin marker was amplified using primers Km3
and Km4. As template for PCR reactions, chromosomal DNA
from B. subtilis 168 was used. The kanamycin marker was
amplified from an aph3 containing plasmid. The PCR fragments were digested, ligated, and the ligation product was
directly transformed to competent 168 cells. An ezrA deletion
(B. subtilis 3362) was constructed by double crossover of a
tetracycline marker. The upstream region was amplified by
PCR using primers nifZ2 and ezrA1, and the downstream
region was amplified using primers ezrA2 and ytrP1. The
tetracycline marker was cut out of pBEST309, ligated with
the upstream and downstream fragments, and the ligation
product was directly transformed to competent B. subtilis 168
cells. B. subtilis 4055 was constructed as follows. The ylmBC region plus the ylmD promoter region was replaced with
the pMUTIN4 derivative pMut by double crossover. pMut is
pMUTIN4 without the lacZ gene, and was derived by PCR
reaction on pMUTIN4 with primers pmut1 and pmut2. The
ylmB-C upstream region was amplified by primers sigE1 and
ylmA2, and the downstream region was amplified by primers
Fluorescence light microscopy
Cells from overnight cultures grown in PAB were inoculated
into fresh PAB. The following B. subtilis strains required 1 mM
IPTG for growth: 4077, 4216, 4217, 4218 and 4219.
B. subtilis 4219 also required 0.5% xylose. To induce the
sepF-gfp, gfp-ftsZ and gfp-pbp2B fusions, 0.1%, 0.2% and
0.5% xylose was used respectively. Depletion of FtsZ, FtsL,
Pbp2B or SepF was achieved by growing exponentially growing cultures for 2 h in the absence of IPTG. For fluorescence
microscopy, cultures were grown in PAB at 30°C, samples
were taken at exponentially growth, and mounted onto microscope slides coated with a thin layer of 1.5% agarose.
Images were acquired with a Zeiss Axiovert 200M coupled
to a CoolsnapHQ CCD camera, and using Metamorph imaging software (Universal Imaging). When required, cells were
grown in the presence of the membrane dye FM5-95
(90 µg ml−1, Molecular Probes). For cell length measurements, cells were mixed with the membrane dye Nile Red
(2.5 µg ml−1, Molecular Probes), prior to microscopic
examinations.
Electron microscopy
For transmission electron microscopy (TEM), cultures were
grown in PAB at 37°C to mid-exponential phase and fixed by
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999
998 L. W. Hamoen et al.
the addition of gluteraldehyde to a final concentration of
2.5%. Cells were then pelleted and fixation was continued
overnight at 4°C. Cell pellets were washed with 200 mM
phosphate buffer, post fixed with 1% osmium tetroxide in
100 mM phosphate buffer, and incubated for 1 h at 4°C. Pellets were then washed in distilled water and stained with 2%
aqueous uranyl acetate for 1 h at 4°C in the dark. The fixed
cells were washed twice in water, dehydrated in acetone, and
washed in propylene dioxide. Samples were imbedded in
epon-araldite resin that was allowed to polymerize overnight
at 65°C. Sections of 80 nm were cut using an ultracut microtome (Reichert and Jung), and examined with a Zeiss EM
912 OMEGA Electron Microscope.
Northern blotting
Total RNA was isolated from exponentially growing cells
using the FastRNA Pro-kit (Qbiogene). For Northern blotting,
8 µg of RNA was denaturated using 10 µl denaturation-mix
(10× FA buffer, 37% formaldehyde and formamide;
20:34:100) at 55°C for 15 min. 10× FA buffer contains
200 mM MOPS (pH 7), 50 mM Na-acetate and 10 mM EDTA.
RNA was separated on formaldehyde agarose gels (0.5 g
agarose, 4 ml 10× FA buffer, 720 µl 37% formaldehyde, in
40 ml), run in 1× FA buffer (100 ml 10× FA buffer and 20 ml
37% formaldehyde, in 1 l), and blotted onto a nylon membrane (Amersham) using 20× SSC blot buffer (3 M NaCl,
0.3 M Na-citrate pH 7). RNA was immobilized at 55°C for 3 h,
and hybridized with radioactively labelled probes directed
against ylmD, ylmG, ylmH and divIVA. Probes were synthesized using the Prime-a-gene labelling kit (Promega). Blots
were hybridized at 67°C and subsequently exposed to a
phosphor screen.
Yeast two-hybrid assay
The different proteins that have been tested for interaction in
the yeast two-hybrid assay are listed in the Results section.
The related open reading frames were PCR amplified from
chromosomal DNA, and PCR products were cloned into
pGBDU (bait) and pGAD (prey) vectors (James et al., 1996)
using the EcoRI and BamHI restriction sites. The resulting
vectors were checked by DNA sequencing, and the yeast
strains PJ69–4a (bait) and PJ69–4α (prey) (James et al.,
1996) were transformed by the bait and prey vectors respectively. Bait-containing cells were selected on SC-U medium
(Synthetic Complete lacking uracil), and prey-containing cells
were selected on SC-L medium (Synthetic Complete lacking
leucine). For each construct, two independent transformants
were stored for further mating experiments. Prey- and baitcontaining strains were grown on fresh selective plates at
30°C for 48 h, and cells were resuspended in 5 ml YEPD
liquid medium. Mating was carried out by mixing 50 µl of
prey- and bait-containing cells in 96 well plates, and depositing, with a replicating tool, approximately 3 µl of the cell
mixtures (prey + bait) onto YEPD plates, which were incubated at 30°C for 48 h. Cells were transferred onto SC-LU
medium (Synthetic Complete lacking leucine and uracil) for
diploid selection, and the plates were incubated for 2–3 days
at 30°C. Diploid colonies were then transferred onto media
selecting for the expression of the HIS3 and ADE2 interaction
reporters (SC-LUH and SC-LUA; Synthetic Complete lacking
leucine, uracil and histidine or adenine respectively). Interaction phenotypes were scored after 5 days of growth at 30°C.
Acknowledgements
We would like to thank the members of the group for helpful
advice and discussions, and Michael Shaw for expert help
with the EM studies, and Naotake Ogasawara for communicating results prior to publication. This research was supported by an EMBO Long-Term Fellowship (LH), a grant from
the UK Biotechnology and Biological Science Research
Council (JE), and a fellowship from the EPA Cephalosporin
Fund and INRA (JM).
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Supplementary material
The following supplementary material is available for this
article online:
Fig. S1. Comparison of the ylm locus in different Gram positive bacteria. The conserved ftsA, ftsZ and ileS genes are
also indicated. The B. subtilis ylm locus is located approximately 8 kb downstream of ftsZ. divIVA is indicated in black,
and the conserved ylm genes (ylmE, -F, -G and -H) are
marked with different shades of grey.
Table S1. Primers used for PCR reactions.
This material is available as part of the online article from
http://www.blackwell-synergy.com
© 2005 The Authors
Journal compilation © 2005 Blackwell Publishing Ltd, Molecular Microbiology, 59, 989–999