University of Groningen
Spo0A regulates chromosome copy number during sporulation by directly binding to
the origin of replication in Bacillus subtilis
Boonstra, Mirjam; de Jong, Imke G.; Scholefield, Graham; Murray, Heath; Kuipers, Oscar;
Veening, Jan-Willem
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Molecular Microbiology
DOI:
10.1111/mmi.12141
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Boonstra, M., de Jong, I. G., Scholefield, G., Murray, H., Kuipers, O. P., & Veening, J-W. (2013). Spo0A
regulates chromosome copy number during sporulation by directly binding to the origin of replication in
Bacillus subtilis. Molecular Microbiology, 87(4), 925-938. DOI: 10.1111/mmi.12141
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Molecular Microbiology (2013) 87(4), 925–938 䊏
doi:10.1111/mmi.12141
First published online 21 January 2013
Spo0A regulates chromosome copy number during
sporulation by directly binding to the origin of replication in
Bacillus subtilis†
Mirjam Boonstra,1‡ Imke G. de Jong,1‡
Graham Scholefield,2 Heath Murray,2
Oscar P. Kuipers1,3* and Jan-Willem Veening1**
1
Molecular Genetics Group, Groningen Biomolecular
Sciences and Biotechnology Institute, Centre for
Synthetic Biology, University of Groningen, Nijenborgh
7, 9747 AG, Groningen, the Netherlands.
2
Centre for Bacterial Cell Biology, Institute for Cell and
Molecular Biosciences, Newcastle University, Newcastle
Upon Tyne NE2 4AX, UK.
3
Kluyver Center for Genomics of Industrial
Fermentation, Groningen/Delft, Netherlands Genomics
Initiative/Netherlands Organization for Scientific
Research, the Netherlands.
Summary
When starved, Bacillus subtilis cells can enter the
developmental programme of endospore formation
by activation of the master transcriptional regulator
Spo0A. Correct chromosome copy number is crucial
for the production of mature and fully resistant
spores. The production and maintenance of one chromosome for the mother cell and one copy for the
forespore requires accurate co-ordination between
DNA replication and initiation of sporulation. Here,
we show that Spo0A regulates chromosome copy
number by directly binding to a number of Spo0A
binding sites that are present near the origin of replication (oriC). We demonstrate that cells lacking three
specific Spo0A binding sites at oriC display increased
chromosome copy numbers when sporulation is
induced. Our data support the hypothesis that Spo0A
directly controls DNA replication during sporulation
by binding to oriC.
Accepted 20 December, 2012. For correspondence. *E-mail
[email protected]; Tel. (+31) (0)50 363 2093; Fax (+31) (0)50 363
2348; **E-mail [email protected]; Tel. (+31) (0)50 363 2408; Fax
(+31) (0)50 363 2348. †This article is dedicated to the memory of
Henny Kalk. ‡These authors contributed equally.
© 2013 Blackwell Publishing Ltd
Introduction
Bacillus subtilis is widely used as a model organism for
studying cell cycle progression and cellular differentiation.
Under growth promoting conditions, B. subtilis divides
symmetrically, giving rise to two identical cell types. When
exposed to stress, such as nutrient starvation, a subpopulation of cells initiates sporulation. Sporulation is a costly
and time-consuming process resulting in the development
of two different cell types: the larger mother cell and the
smaller forespore. Importantly, both cell types inherit a
single copy of the chromosome during sporulation (Fig. 1)
(Errington, 2003; Higgins and Dworkin, 2012). Tight
co-ordination between DNA replication and sporulation
results in correct chromosome copy number, and is a
requirement for efficient sporulation (Murray and Errington,
2008; Eldar et al., 2009; Veening et al., 2009; Xenopoulos
and Piggot, 2011). So far, two checkpoint factors have
been identified that play a role in this: SirA (sporulation
inhibitor of replication A) and Sda (suppressor of dnaA1).
SirA prevents initiation of DNA replication in cells committed to sporulation. Transcription of sirA is directly activated
by Spo0A~P, the master regulator of sporulation. SirA
maintains the diploid state of sporulating cells by directly
targeting the DNA replication initiator protein DnaA which
consequently becomes displaced from the origin of replication (oriC) (Rahn-Lee et al., 2009; 2011; Wagner et al.,
2009). While SirA prevents re-initiation of DNA replication
in sporulating cells, the checkpoint function of Sda is to
prevent sporulation initiation in replicating cells and during
DNA repair (Burkholder et al., 2001; Veening et al., 2009).
The intrinsically unstable Sda protein binds to the major
sporulation kinases (KinA and KinB) and prevents their
autophosphorylation (Burkholder et al., 2001; Rowland
et al., 2004; Ruvolo et al., 2006; Whitten et al., 2007; Cunningham and Burkholder, 2009). KinA and KinB are proteins of the so-called phosphorelay, which regulates the
level of phosphorylated Spo0A (the transcriptionally active,
DNA-binding form of Spo0A) (Burbulys et al., 1991). Interestingly, pulses of Sda synthesis occur concomitantly with
the initiation of DNA replication via transcriptional activation by DnaA. Following the burst of Sda expression,
926 M. Boonstra et al. 䊏
Fig. 1. Relation between DNA replication and sporulation. During sporulation, one copy of the chromosome is pumped in the forespore and
one copy of the chromosome remains in the mother cell. It should be noted that during rapid growth, B. subtilis keeps at least two copies of
the chromosome per cell.
targeted proteolysis of Sda reduces its concentration to
generate a window of opportunity at the end of the replication cycle during which cells can enter sporulation as
diploids. Cells that do not enter sporulation during that
small time period have to undergo a new round of replication before getting the next opportunity to do so (Veening
et al., 2009). Mother cells and endospores of a sda/sirA
double mutant frequently contain more than one copy of
the chromosome and polyploid spores show reduced
fitness (Veening et al., 2009). Relevant for the current
study is the fact that even in the sda/sirA double mutant, the
majority of sporulating cells still show wild-type chromosome copy numbers and wild-type fitness. Hence, additional factors are likely at play to co-ordinate DNA
replication with sporulation.
In order to initiate DNA replication, DnaA binds various
perfect and imperfect DnaA-boxes (aA-boxes) present
within the oriC region where it assembles into a large
nucleoprotein complex. This results in denaturation of an
AT-rich region at oriC, which is required for the assembly
of a functional replisome (Moriya et al., 1999; Messer,
2002). Interestingly, Castilla-Llorente et al. (2006) noted
that the aA-box sequence (consensus 5′-TGTGNATAA-3′;
Fig. 2, orange) partially overlaps with the recognition
sequence for Spo0A (the 0A-box, consensus 5′TGTCGAA-3′; Fig. 2, blue, Strauch et al., 1990; Molle
et al., 2003), and indeed the B. subtilis origin region contains many Spo0A binding sites that are specifically
recognized by purified protein in vitro (Fig. 2; Castilla-
Llorente et al., 2006). However, it remains unclear
whether the binding of Spo0A at the B. subtilis origin is
physiologically relevant for B. subtilis, as studies performed to show a possible direct effect of Spo0A on DNA
replication have not led to conclusive results. On the one
hand it was shown that Spo0A can inhibit DnaAdependent DNA duplex unwinding in vitro (Fig. 2; CastillaLlorente et al., 2006). On the other hand Spo0A does not
seem to have a significant effect on DNA replication in
vivo when sporulation is artificially induced in exponentially growing cells (Wagner et al., 2009). Spo0A~P also
reaches high levels in the mother cell (Fujita and Losick,
2003) and recently the Piggot lab showed that Spo0A~P
inhibits mother cell growth and DNA replication independently of SirA. The molecular mechanism for this Spo0Adependent repression of replication is yet unknown,
although a possible role for Sda in this has been suggested (Xenopoulos and Piggot, 2011).
Here, we have investigated the role of Spo0A as a
direct inhibitor of DNA replication initiation during sporulation by mutating the 0A-boxes within oriC, which is the
most decisive method to assess a possible direct role of
Spo0A in the control of DNA replication. The results indicate that Spo0A directly controls chromosome copy
number by binding to a number of specific Spo0A
binding sites present within the oriC region and that this
regulation is especially important in the absence of SirA
and Sda or when sporulation is induced in actively replicating cells.
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
Spo0A controls DNA replication 927
Fig. 2. A. Schematic representation of the origin of replication (oriC) of Bacillus subtilis showing approximate positions of Spo0A- and
DnaA-boxes (0A- and aA-boxes) within the incA, incB and incC regions (Fukuoka et al., 1990; Castilla-Llorente et al., 2006). The 0A-box
(consensus sequence 5′-TGTCGAA-3′) partially overlaps with the inverted aA-box (consensus sequence 5′-TTATNCACA-3′, inverted
consensus sequence 5′-TGTGNATAA-3′). The promoter of DnaA lies in between aA-box 6 and 7 (Ogasawara et al., 1985) and its approximate
position is indicated by an arrow. Note that the graph is not drawn to scale.
B. Sequence of the native oriC (oriCnat).
C. Sequence of the mutated oriC (oriCmut).
0A-boxes are indicated in blue, aA-boxes in orange. The lighter the colour, the less similar the sequence is to its consensus. Arrows indicate
the direction of these recognition sequences on the chromosome. Nucleotides shown in bold are used to introduce point mutations. ‘X‘
indicates mismatches compared with the consensus sequences of Spo0A- and DnaA-boxes. Red ‘X‘ indicates mismatches introduced by
mutation.
Results
Mutating 0A-boxes within the origin of replication
The B. subtilis oriC region contains several bona fide
Spo0A binding sites (consensus sequence 5′-TGTCGAA3′; Fig. 2A, blue) that partially overlap with functional DnaA
binding sites (inverted consensus 5′-TGTGNATAA-3′;
Fig. 2A, orange, Castilla-Llorente et al., 2006). If Spo0A
interferes with DNA replication initiation in vivo, then sporulating cells without functional Spo0A binding sites in the
oriC region are expected to over-replicate. To test this
hypothesis, we mutated the three 0A-boxes within the oriC
region that were shown to bind Spo0A in vitro with high
affinity (Castilla-Llorente et al., 2006) (0A-boxes 3, 5 and 8;
Fig. 2B and C). The 0A-boxes were altered in such a way
that the number of mismatches of the corresponding
0A-box to its consensus was increased, whereas the
number of mismatches of the corresponding overlapping
aA-box in relation to the aA-box-consensus was not altered
(aA-boxes 10 and 12) or reduced (aA-box 3) (compare the
bold bases, which highlight the bases used for mutation,
and the ‘X’ which mark the mismatches to the correspond-
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
928 M. Boonstra et al. 䊏
Fig. 3. DnaA binds with similar efficiency to oriCmut compared with oriCnat in vitro. The incAB PCR product (474 bp, 36 nM) was incubated
with twofold increasing concentrations, ranging from 94 nM to 12 mm, of DnaA-ADP (A) or DnaA-ATP (B). Nucleoprotein complexes were
separated on a 1.5% agarose gel and visualized by staining with ethidium bromide.
ing consensus in Fig. 2B with Fig. 2C). In order to diminish
the chance of negatively affecting DnaA binding to the oriC,
existing recognition sequences of DnaA within the oriC
region were used for the design when possible. The wildtype, native oriC region (oriCnat) was altered by allelic
replacement using a synthetic oriC containing the mutated
0A-boxes (oriCmut) to obtain strain IDJ056 (see Experimental procedures). Strain IDJ056 (oriCmut) and its wild-type
parental strain IDJ055 (oriCnat) exhibit similar doubling
times and similar dnaA transcript levels and DnaA protein
levels during steady-state growth (Fig. S1). To test whether
DnaA binding to oriCmut is affected, we purified B. subtilis
DnaA and performed electrophoretic mobility shift assays
(EMSA). Since DnaA can bind to DNA in both ADP- and
ATP-bound forms (Sekimizu et al., 1987), we carried out
EMSAs in the presence of either nucleotide. As shown in
Fig. 3, the purified DnaA protein bound similarly to both the
wild-type and mutant DNA fragments. Taken together,
these results indicate that DnaA-binding efficiency to the
mutated oriC is not significantly altered.
Spo0A binds less efficiently to the mutated oriC region
To test whether Spo0A~P is able to bind oriCmut in vitro, we
purified C-terminally his-tagged Spo0A variants and performed EMSAs. Spo0A consists of two domains, the
C-terminal DNA-binding domain and the N-terminal phosphoacceptor domain. Phosphorylation of a conserved
aspartic acid residue within the N-terminal domain results
in a conformational change of Spo0A~P, upon which the
protein dimerizes and becomes active (Ladds et al., 2003;
Muchová et al., 2004). It has been reported that Spo0A
purified from Escherichia coli is only active up to 40%, while
other studies did not obtain active Spo0A without subsequent in vitro phosphorylation steps (Lewis et al., 2002;
Ladds et al., 2003). Therefore, we also purified the DNAbinding domain of Spo0A alone (Spo0A-DB), which does
not require phosphorylation for DNA binding (Molle et al.,
2003). Full-length Spo0A (Spo0A-FL) and Spo0A-DB were
incubated with [g-33P]-labelled oriC DNA containing the
0A-boxes within incA, incB or both of the incA and incB
regions that harbour the 0A-boxes of interest (Figs 2 and
4A). As a negative control, we used the srfAA promoter and
as a positive control the abrB promoter (Molle et al., 2003).
As shown in Fig. 4B, Spo0A-DB as well as Spo0A-FL bind
the oriCnat DNA fragments at concentrations of 30–120 nM,
whereas no or poor shifts were obtained after incubation
with our synthetic oriCmut DNA, confirming that the introduced mutations are effective and reduce Spo0A binding.
Interestingly, in contrast to Spo0A-FL, incubation of the
oriCnat DNA with Spo0A-DB results in multiple retarded
species. This difference may be due to the fact that
whereas active Spo0A-FL binds DNA as a dimer,
Spo0A-DB binds DNA as a monomer (Lewis et al., 2000).
Therefore, different affinities of individual 0A-boxes may be
reflected in different retarded species. Overall, the results
presented in Figs 3 and 4 show that the mutations introduced and present in oriCmut affect the in vitro affinity of
Spo0A to this region without significantly altering DnaA
binding.
0A-boxes within the oriC function as DNA replication
control elements during sporulation
Next, we set out to investigate the differences in chromosome copy number between cells in which Spo0A binding
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
Spo0A controls DNA replication 929
Fig. 4. Spo0A binds less efficiently to oriCmut compared with oriCnat in vitro.
A. Overview of the PCR fragments used for the EMSAs. Mismatches within the 0A-box are indicated as ‘X‘. Red ‘X‘ indicates mismatches
introduced by mutation. 0A-boxes of interest are shown in blue.
B. Full-length Spo0A (Spo0A-FL) as well as the DNA-binding domain of Spo0A (Spo0A-DB) bind less efficiently to oriC PCR fragments
harbouring mutated 0A-boxes (oriCmut) compared with oriC fragments in which no additional mismatches were introduced (oriCnat). Protein
concentrations: 30, 60, 90 and 120 nM. The promoter region of srfA (PsrfA) was used as a negative control to which Spo0A~P does not bind.
As a positive control, we used the promoter region of abrB (PabrB), to which Spo0A~P binds with high affinity (Molle et al., 2003).
to the oriC is unaffected (oriCnat) or disturbed (oriCmut). In
order to monitor the number of replication events we
integrated an array of tet operator sites close to the oriC (at
345° of the circular chromosome) and a xylose-inducible
tetR-mCherry gene at the non-essential amyE locus in
both strains (oriCnat and oriCmut). Upon xylose induction,
mCherry-labelled TetR binds to the tet operator sites near
oriC, and the number of fluorescent foci will reflect the
number of oriC regions present in the cell (see Veening
et al., 2009, Fig. 5A). We also generated reporter strains
that are mutated for sda, sirA or both sda and sirA to test for
possible additive/synthetic effects of these known checkpoint mechanisms. Since the conditions that induce sporulation are important to observe replication checkpoint
mechanisms (Wagner et al., 2009), we first triggered
sporulation upon gradually depleting nutrients and increasing cell density by growing cells in Schaeffer’s sporulation
medium (Schaeffer et al., 1965). Under these conditions,
cells grow initially fast, similar to cells growing in LB
medium. However, in contrast to LB medium, sporulation is
efficiently initiated in a large subpopulation of cells once
nutrients become limiting and cell densities high.
The TetR-mCherry reporter strains harbouring either
oriCmut or oriCnat in the different genetic backgrounds (wild
type, Dsda, DsirA or Dsda/sirA) were allowed to initiate
sporulation and at least two biological replicates per strain
were taken to determine the number of mCherry foci in the
mother cell compartment of cells containing a phase bright
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
930 M. Boonstra et al. 䊏
Fig. 5. Cells carrying the mutated oriC
(oriCmut) over-initiate replication in the
absence of both the SirA and the Sda
checkpoints when sporulation is induced in
slowly growing cells. Replication initiation
events are monitored by fluorescence
microscopy of sporulating cultures of strains
harbouring a xylose-inducible tetR-mCherry
fusion and tet operator sites near the oriC.
A. Example of cells with different numbers of
oriC-mCherry foci representing variations in
replication initiation events (arrows).
B–E. Number of oriC-mCherry foci in mother
cells of cultures grown in Schaeffer medium
(number of cells in %). Error bars represent
standard errors of the mean. (B) Wild
type (IDJ075, oriCnat; IDJ076, oriCmut),
(C) Dsda (IDJ077, oriCnat; IDJ078, oriCmut),
(D) DsirA (IDJ079, oriCnat; IDJ080, oriCmut),
(E) and Dsda/sirA double mutant (IDJ081,
oriCnat; IDJ082, oriCmut).
F. Comparison of the average numbers of
mCherry foci per cell counted for wild type
(IDJ075, IDJ076), Dsda (IDJ077, IDJ078),
DsirA (IDJ079, IDJ080) and Dsda/sirA double
mutant mother cells (IDJ081, IDJ082) with
oriCnat and oriCmut genetic backgrounds
respectively. Significant differences
(P < 0.001), according to a Mann–Whitney
U-test, are marked with an asterisk.
forespore (Fig. 5B–F). Due to strong autofluorescence of
the phase-bright forespore compartment, it was not possible to detect oriC-mCherry foci in forespores. If replication
and sporulation are co-ordinated correctly, mother cells (as
well as forespores) are expected to contain exactly one
chromosome (Fig. 1). As shown in Fig. 5B and D, wild-type
mother cells (IDJ075, IDJ076) as well as DsirA mother cells
(IDJ079, IDJ080) mainly have one oriC focus, irrespective
of a native or mutated origin of replication. Interestingly,
mother cell compartments with more than one fluorescent
focus were frequently observed in the single sda mutant
containing oriCnat (strain IDJ077) (Fig. 5C and F). Moreover, the fraction of Dsda mother cells containing more than
one fluorescent foci was slightly elevated when it also
contained the oriCmut region (strain IDJ078; average of 1.16
versus 1.18 foci; n > 450) (Fig. 5C and F). However, this
difference is not statistically significant (P = 0.244, Mann–
Whitney U-test). Therefore, the obtained results indicate
that, under the conditions tested, the co-ordination
between DNA replication and sporulation is not significantly affected in oriCmut cells when tested in a wild-type, or
in a single Dsda or DsirA mutant background.
However, the co-ordination between DNA replication
and sporulation was significantly affected in cells containing oriCmut when this was tested in a Dsda/DsirA doublemutant background. The average origin copy number in
mother cells were 1.48 and 1.26 (n > 430 cells; P < 0.001,
Mann–Whitney U-test) in the case of strains IDJ082
(oriCmut) and IDJ081 (oriCnat) respectively (Fig. 5E and F).
These results demonstrate that the presence of bona fide
Spo0A binding sites in the oriC region contributes to proper
co-ordination of DNA replication and sporulation. In addition, the results show that, at least under the conditions
tested, sda is the locus that mostly contributes to proper
co-ordination between replication and sporulation since no
significant defect in this co-ordination is observed in a sirA
or oriCmut strain as long as sda is not affected.
Induction of sporulation in actively replicating cells
leads to increased chromosome copy numbers in
oriCmut strains
In the previous section, the effect of oriCmut was tested
under conditions in which sporulation is rather gradually
induced upon starvation and high cell density. However, in
nature the growth conditions can change rapidly, and
apparently redundant mechanisms may contribute differently under different conditions, as also suggested by
Xenopoulos and Piggot (2011). Therefore, we examined
functionality of Spo0A binding sites within oriC when sporulation was induced in actively growing and replicating cells,
which was achieved by conditionally inducing kinA from an
ectopic locus. KinA is the major sporulation kinase and
induction of KinA in exponentially growing and actively
replicating cells initiates sporulation by gradually increasing the intracellular levels of Spo0A~P (Fujita and Losick,
2005). The kinA gene was placed under control of the IPTG
(isopropyl-b-D-thiogalactopyranoside)-inducible promoter
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
Spo0A controls DNA replication 931
Fig. 6. Initiation of sporulation by kinA induction in fast-growing cells results in an increased chromosome copy number when Spo0A binding
at oriC is reduced in vegetative cells. Cells were grown in the presence of 1 mM IPTG and harvested for fluorescence microscopy analysis at
early-exponential growth after 3 h of induction (from the start of inoculation).
A. Wild type (MB003, Pspank-kinA, oriCnat; MB004, Pspank-kinA, oriCmut).
B. Dsda (MB005, Pspank-kinA, oriCnat; MB006, Pspank-kinA, oriCmut).
C. DsirA (MB007, Pspank-kinA, oriCnat; MB008, Pspank-kinA, oriCmut).
D. Dsda/sirA (MB009, Pspank-kinA, oriCnat; MB010, Pspank-kinA, oriCmut).
The error bars represent the standard deviation. The difference in oriC copy number is significant for all strains (Mann–Whitney U-test)
(Table 1). Pie charts show the fraction of cells with a certain number of origins.
Pspank, and the resulting construct was introduced into the
oriCnat and oriCmut TetR-mCherry strains. In addition, PspankkinA in the oriCmut and oriCnat background was combined
with single or double mutation in sda and sirA. The resulting
strains, MB003–MB010, were grown in Schaeffer’s sporulation medium containing 1 mM IPTG and samples were
taken for fluorescence microscopy during exponential
growth and during sporulation when phase bright forespores became visible. Strikingly, independent of the
genetic background tested, induction of sporulation in
exponentially growing cells caused a significant increase in
fluorescent foci when they contained oriCmut (P < 0.005
Mann–Whitney U-test, n > 470, in all cases tested). Differences were observed both in exponentially growing cells
and in mother cells during sporulation (Figs 6 and 7 and
Table 1). These results show that initiation of sporulation in
actively replicating cells significantly affects chromosome
copy number when the 0A-boxes are removed, even in the
presence of the SirA and Sda checkpoints.
It is interesting to note that under these conditions the
defects in proper copy number regulation in cells containing oriCmut becomes particularly evident in the DsirA
mutant background (on average 1.07 versus 1.45 oriC
foci per mother cell in DsirA/Pspank-kinA; Table 1). Whereas
the results presented above showed that Sda was most
prominent in the proper control of mother cell copy
number when sporulation is induced as a consequence of
nutrient starvation and high cell density, the latter results
indicate that SirA plays a particularly important role in
DNA replication control when sporulation is initiated in
actively growing cells, as was shown previously (Wagner
et al., 2009).
To determine whether the effect of kinA overexpression
on oriC copy numbers is indeed the result of increased
levels of Spo0A~P, spo0A was deleted in the oriCnat
(MB003) and oriCmut (MB004) Pspank-kinA strains. Cultures
were induced with 1 mM IPTG upon inoculation in Schaeffer’s sporulation medium and samples were taken only
during mid-exponential growth, since cells lacking spo0A
do not sporulate (Hoch, 1976). We found no significant
difference between the oriCnat and oriCmut Pspank-kinA
strains in the absence of spo0A (Fig. 8), demonstrating
that the effect of KinA overproduction on replication
depends on Spo0A~P and is not an artefact of kinA
overexpression.
In summary, our data provide strong evidence that,
especially when sporulation is induced in fast-growing
cells, Spo0A~P contributes significantly in the control of
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
932 M. Boonstra et al. 䊏
Fig. 7. Initiation of sporulation by kinA induction results in an increased chromosome copy number when Spo0A binding at oriC is reduced in
mother cells. Cells were induced at mid-exponential growth phase with 1 mM IPTG and harvested for fluorescence microscopy analysis in
stationary phase after 4 h of IPTG induction when the first phase bright spores became visible.
A. Wild type (MB003, Pspank-kinA, oriCnat; MB004, Pspank-kinA, oriCmut).
B. Dsda (MB005, Pspank-kinA, oriCnat; MB006, Pspank-kinA, oriCmut).
C. DsirA (MB007, Pspank-kinA, oriCnat; MB008, Pspank-kinA, oriCmut).
D. Dsda/sirA (MB009, Pspank-kinA, oriCnat; MB010, Pspank-kinA, oriCmut).
The error bars represent the standard deviation. The difference in oriC copy number is significant for all strains (Mann–Whitney U-test)
(Table 1). Pie charts show the fraction of cells with a certain number of origins.
DNA replication initiation and hence proper co-ordination
with sporulation by binding to its cognate binding sites in
the oriC region.
Discussion
Table 1. The fraction of cells with abnormal chromosome copy
numbers increases in the absence of specific Spo0A binding sites
at oriC.
A
Average oriC number
Exponential
Spo0A, the master regulator of sporulation, has been
shown to indirectly inhibit DNA replication by regulating
genes involved in replication and cell division (Molle et al.,
2003; Rahn-Lee et al., 2009; 2011; Wagner et al., 2009).
Whether Spo0A also directly regulates DNA replication of
B. subtilis in vivo is debated in the literature (see, e.g.
Molle et al., 2003; Castilla-Llorente et al., 2006; Wagner
et al., 2009; Xenopoulos and Piggot, 2011). For instance,
induction of a constitutively active allele of Spo0A (called
Spo0A-sad67; Ireton et al., 1993) in the B. subtilis PY79
genetic background showed no downregulation of initiation of DNA replication in the absence of SirA, indicating
that SirA rather than Spo0A is the major factor controlling
DNA replication under these experimental conditions
(Wagner et al., 2009). However, Spo0A-sad67 is highly
toxic to the cells (Ireton et al., 1993) and the sad67 allele
was also shown to be unstable in the presence of a
oriCnat
oriCmut
P*
Wild-type Pspank-kinA
Dsda Pspank-kinA
DsirA Pspank-kinA
Dsda/sirA Pspank-kinA
2.42
2.60
2.51
2.66
2.58
2.94
2.75
2.80
< 0.001
< 0.001
< 0.001
< 0.001
B
Average oriC number
Mother cells
Wild-type Pspank-kinA
Dsda Pspank-kinA
DsirA Pspank-kinA
Dsda/sirA Pspank-kinA
oriCnat
oriCmut
P*
1.03
1.05
1.07
1.31
1.10
1.09
1.45
1.71
< 0.001
0.005
< 0.001
< 0.001
*Significance was calculated using a Mann–Whitney U-test.
(A) Number of oriC foci in exponentially growing or in mother cells (B)
of cultures induced to produce KinA.
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
Spo0A controls DNA replication 933
Fig. 8. Induction of sporulation by Pspank-kinA in a Dspo0A
background does not result in a significant oriC copy number
difference between oriCnat and oriCmut strains. Cells were grown
with 1 mM IPTG and collected for fluorescence microscopy at
mid-exponential growth (note that the cells analysed for Fig. 6 were
from early-exponential growth phase).
A. Comparison of the oriC number of the Dspo0A, Pspank-kinA,
oriCnat (MB016) and Dspo0A, Pspank-kinA oriCmut (MB017) strains.
B. The difference in oriC numbers between MB016 and MB017 is
not significant (P = 0.379, Mann–Whitney U-test).
DnaX–YFP fusion indicating pleiotropic consequences
of its induction (Wagner et al., 2009). Furthermore, as
shown here, the growth conditions and timing of sporulation initiation relative to replication status appear to be
crucial to observe the direct effects of Spo0A~P on chromosome copy number (Figs 5–7).
The oriC of B. subtilis harbours a number of bona fide
Spo0A binding sites that overlap with functional DnaA
binding sites (Fig. 2, blue), and binding of Spo0A to these
sequences has been shown in vitro (Castilla-Llorente
et al., 2006). In addition, Castilla-Llorente et al. showed
that Spo0A can inhibit DnaA-mediated open complex formation in vitro (Castilla-Llorente et al., 2006). Interestingly,
they also showed that Spo0A binds the origins of the
virulent B. subtilis phage F29 in vivo, thereby preventing
amplification of F29 DNA after infection which forces the
phage genome to remain dormant in cells with active
Spo0A (Castilla-Llorente et al., 2006). In F29, binding of
Spo0A~P prevents formation of the replication initiation
nucleoprotein complex by preventing and displacing the
replication initiator protein p6 from the origins (CastillaLlorente et al., 2006). Inspired by these findings, we
examined whether B. subtilis Spo0A~P also affects DNA
replication in vivo by constructing strains that lack the
particular 0A-boxes to which Spo0A~P binds with high
affinity within the oriC region (Fig. 2C and Castilla-Llorente
et al., 2006). Electrophoretic mobility shift assays confirmed that Spo0A binds less efficiently to our mutated
oriCmut compared with the native oriCnat in vitro, while DnaA
still binds at near wild-type efficiency (Figs 3 and 4).
To investigate a possible direct effect of Spo0A on replication in vivo, we monitored origin copy number by means
of imaging TetR-mCherry foci that localize near the oriC
(Fig. 5A). Interestingly, we found that the contribution of
Spo0A~P in proper co-ordination of replication and sporulation varies upon the growth conditions that induce sporulation. A rather small effect of oriC-located Spo0A binding
sites on DNA replication control was observed when sporulation was induced in cells in which growth has been
slowed down, which happens during nutrient depletion
in Schaeffers medium at the end of the exponential
growth phase. Under these conditions, the effect was only
significant in the Dsda/DsirA double mutant background
(Fig. 5E). These results imply that under these conditions,
both SirA and Sda are sufficient to ensure proper regulation
of DNA replication in sporulating cells. The observation that
proper control of replication is lost in Dsda, but not DsirA
single mutant cells indicates that Sda is the most important
locus for co-ordinating replication and sporulation under
these conditions.
Strikingly, the role of Spo0A~P in properly co-ordinating
DNA replication and sporulation is much more pronounced
when rapidly growing cells were suddenly induced to
sporulate. Under these conditions, oriCmut has significant
effects on the regulation of DNA replication even in an
otherwise wild-type background (Figs 6 and 7 and
Table 1). Interestingly, whereas Sda turned out to be most
important when sporulation was induced in cells having
slowed down their growth, SirA appeared most important
when sporulation was induced in rapidly growing cells in
the oriCmut background.
The fraction of cells with increased numbers of oriC foci
in wild-type mother cells is only slightly (but significantly)
elevated in the absence of SirA upon KinA induction in
Schaeffers medium in our genetic JH642 background
(Table 1). Wagner et al. reported a much larger impact of
the absence of SirA when KinA was induced using the
Phyper-spank promoter (which is stronger than the here
used Pspank) in LB medium in the PY79 genetic background, and more than 54% of sporulating cells had more
than two foci (Wagner et al., 2009). Besides the genetic
and experimental differences between the two studies, it
should also be noted that chromosome copy number was
assessed at an earlier stage of sporulation (when the
asymmetric septum became visible using a membrane
stain) thus also accounting for chromosomes destined for
the forespore compartment, while we assessed only the
chromosome copy number in the mother cell when the
forespore was already phase bright.
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
934 M. Boonstra et al. 䊏
Importantly, deletion of spo0A in our strains that overexpress kinA did not result in a significant difference
between oriCnat and oriCmut, indicating that the observed
effect is not an artefact of KinA overproduction (Fig. 8).
Together, our results suggest that Spo0A directly inhibits
initiation of DNA replication by binding to the origin of
replication. In addition, these findings may also explain
the recently observed Spo0A-dependent inhibition of
growth and DNA replication in mother cells (Xenopoulos
and Piggot, 2011).
It is interesting to note that regulation of DNA replication by two-component response regulators, such as
Spo0A, is not unique and might be a more common
mechanism in bacteria. For instance, in the asymmetrically dividing a-proteobacterium Caulobacter crescentus,
DNA replication is controlled by a two-component
response regulator called CtrA to ensure correct coordination between DNA replication and cell differentiation. It has been proposed that CtrA competes with
C. crescentus DnaA for binding to the origin and in that
way regulates chromosomal replication (for a recent
review see Collier, 2012). The mechanism by which
B. subtilis Spo0A directly inhibits DNA replication still
remains to be elucidated. It has been shown in vitro that
Spo0A prevents DnaA-dependent open complex formation within incC (Castilla-Llorente et al., 2006). This might
be caused either by directly impeding DnaA binding to the
oriC sequences where Spo0A is bound, or by altering the
ability of DnaA to assemble into its active ATP-dependent
nucleoprotein complex. It is noteworthy to mention that
the region of the origin with the highest DnaA affinity
(in the incB region; Moriya et al., 1988; Fukuoka et al.,
1990) also contains four consecutive 0A-boxes, of which
one is a perfect match to the consensus sequence.
However, Krause et al. (1997) have indicated, using a
plasmid system, that DnaA-dependent open complex formation can still take place in the absence of the incA and
incB regions (Krause et al., 1997), thus, clearly more
research is required to establish the underlying molecular
mechanism.
In nature, cells encounter ever fluctuating conditions in
which growth can be rapidly inhibited and thereby rapidly
inducing sporulation. Consequently, we speculate that in
their natural environment, B. subtilis cells encounter
fitness disadvantages if the regulatory pathway of Spo0A
binding to the oriC is defective since under rapidly changing conditions Spo0A plays a significant role in coordinating replication with sporulation (Figs 6 and 7,
Table 1). Interestingly, even in the most extreme case
investigated in this study (Pspank-kinA in the sda/sirA double
mutant harbouring oriCmut), 54% of cells were seemingly
still able to correctly co-ordinate DNA replication with
sporulation (Fig. 7D). This suggests that additional, yet
unknown factors contribute to the co-ordination of DNA
replication with sporulation. For B. subtilis it is known that
the nutritional status also controls replication (Wang et al.,
2007). This occurs by the production of the small nucleotides ppGpp and pppGpp upon nutrient starvation. These
so-called ‘alarmones’ directly inhibit primase, an essential
component of the replication machinery (Wang et al.,
2007). This control might indirectly be responsible for
correct copy number control during sporulation. Whether
this or other mechanisms are at play remains to be
investigated.
Experimental procedures
Oligonucleotides, plasmids, strains and media
Oligonucleotides, plasmids and bacterial strains are listed in
Tables S1, S2 and S3 respectively. B. subtilis strains were
grown in TY or Schaeffer medium at 37°C (see below). E. coli
DH5a was used as host for cloning and grown in TY medium
at 37°C. When required, the growth media were supplemented with antibiotics at the following concentrations: 5 mg
ml-1 chloramphenicol, 5 mg ml-1 kanamycin, 100 mg ml-1 spectinomycin, 6 mg ml-1 tetracycline, 0.5 mg ml-1 erythromycin +
12.5 mg ml-1 lincomycin (MLS) for B. subtilis and 100 mg ml-1
ampicillin or 150 mg ml-1 erythromycin for E. coli. Agar (1.5%)
was included for solid medium.
Recombinant DNA techniques and oligonucleotides
Procedures for DNA purification, restriction, ligation, agarose
gel electrophoresis and transformation of E. coli were carried
out as described before (Sambrook et al., 1989). Restriction
enzymes were obtained from Roche (Mannheim, GER) and
all PCRs were performed with Phusion (NEB, UK), unless
stated otherwise. Oligonucleotides were purchased from
Biolegio (Nijmegen, the Netherlands). All constructs were
sequence-verified. B. subtilis was transformed as described
before (Harwood and Cutting, 1990).
Strain construction
IDJ055, in which a chloramphenicol cassette is introduced
between the rpmH gene and the dnaA promoter, was obtained
as follows: the upstream region and downstream region of the
oriC sequence of interest were amplified using B. subtilis 168
chromosomal DNA as a template in combination with
prIDJ241 + prIDJ242 and prIDJ245 + prIDJ246. The chloramphenicol cassette was amplified from pUC19-c with
prIDJ243 + prIDJ244 and inserted between the upstream and
downstream region of the oriC via BamHI and NheI. Transformation of the ligation product to B. subtilis 168 resulted in
strain IDJ055. IDJ056 was obtained as follows: the upstream
region of the oriC region of interest and the chloramphenicol
resistance cassette were amplified from IDJ055 with
prIDJ233 + prIDJ243. Plasmid pIDJ070 harbouring the oriC
region with the mutated 0A-boxes was synthesized by Mr
Gene (Regensburg, Germany) and the 0A-boxes of interest
were amplified from pIDJ070 with prIDJ245 and prIDJ274.
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
Spo0A controls DNA replication 935
Both PCR fragments were ligated via NheI and transformed to
B. subtilis 168 resulting in strain IDJ056. Strains IDJ075,
IDJ077, IDJ079 and IDJ081 were obtained by transforming
JWV212, JWV213, JWV214 and JWV215 with chromosomal
DNA of IDJ055 and selecting for chloramphenicol resistance.
Strains IDJ076, IDJ078, IDJ080 and IDJ082 were obtained by
transforming JWV212, JWV213, JWV214 and JWV215 with
chromosomal DNA of IDJ056 and selecting for chloramphenicol resistance. Correct integration was confirmed by PCR and
integrated DNA was sequence verified in all cases.
To construct plasmid pMB001, which contains Pspank-kinA
and thrC flanking regions, the lacI-Pspank-kinA genes were
cut from plasmid pDR110-kinA (de Jong et al., 2010) using
BamHI and EcoRI and ligated into the corresponding sites
of pDG1664 (Guérout-Fleury et al., 1996). Strains MB003,
MB004, MB005, MB006, MB007, MB009 and MB010 were
created by transformation of IDJ075, IDJ076, IDJ077, IDJ078,
IDJ079, IDJ080, IDJ081 and IDJ082, respectively, with
plasmid pMB001 and selecting for MLS resistance. Correct
integration of Pspank-kinA into the thrC locus was verified by
PCR.
MB015 was created by amplification of the upstream region
and downstream region of spo0A with prIDJ179 + prMB017
and prIDJ182 + prMB018, respectively, using chromosomal
DNA of strain B. subtilis 168 as template. The up- and downstream regions of spo0A and pBEST309 (Itaya, 1992) were
digested with BamHI and ligated. The ligation mixture was
transformed directly to B. subtilis 168 and transformants were
selected on tetracycline. Strains MB016 and MB017 were
created by transformation with MB015 genomic DNA. MB017
was sequenced at the oriC region to confirm that the mutations
remained unaltered.
EMSAs
Spo0A-FL and Spo0A-DB (as C-terminal His6 fusions) were
used directly after purification without additional phosphorylation treatment. All primer sequences used for the PCR products are listed in Table S1. The incA region was amplified
with primers prIDJ247 and prIDJ300; the incB region with
prIDJ276 and prIDJ301; the incA-incB region with prIDJ247
and prIDJ276; PabrB with prIDJ004 and prIDJ281; and PsrfA with
prIDJ282 and prIDJ283. The corresponding fragments were
labelled with [g-33P]-ATP using T4 polynucleotide kinase (Fermentas). Various protein concentrations were incubated at
30°C for 20 min in the following reaction mix: 5000 cpm
labelled DNA, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM
EDTA, 1 mM DTT, 5% glycerol, 0.5 mg ml-1 BSA. Fifteen
microlitres of the mix was loaded onto a native polyacrylamide
gel [4% (v/v) polyacrylamide, 1¥ TBE buffer, 0.125% (w/v)
APS, 0.0125% (v/v) TEMED] and electrophoresed in 1¥ TBE
buffer for 50 min at 100 V. Radioactive signals were detected
at room temperature by autoradiography using Cyclone PhosphorImager (Packard Instruments, Meridien, CT) and OptiQuant analysis software (Packard Instruments, Meridien, CT)
after overnight incubation on phosphoscreens.
The EMSAs with DnaA were performed as previously
described (Scholefield et al., 2012). In brief, the DnaA protein
was serially diluted in twofold steps from 12 mm to 94 nM and
incubated with either ATP or ADP (2 mM). The incAB PCR
product (474 bp) was created with primers oGJS61/oGJS62,
purified using a Qiagen PCR purification kit, and utilized at
36 nM. Nucleoprotein complexes were separated on a 1.5%
agarose gel and visualized by staining with ethidium bromide.
RT-QPCR
Spo0A and DnaA purification
Escherichia coli BL21 containing either pMF14 (Molle et al.,
2003) or pETspo0AHIS (Fujita and Losick, 2003) were grown
at 30°C in LB medium and induced with 1 mM IPTG for
overproduction of full-length Spo0A (Spo0A-FL) or the DNAbinding domain of Spo0A (Spo0A-DB) at OD600 = 0.6. Cells
were harvested 5 h after induction (10.000 rcf, 10 min, RT) and
the cell pellets were stored at -80°C. After resuspension of the
pellet in wash buffer (300 mM NaCl, 50 mM NaH2PO4 pH 8.0,
10 mM imidazole), the cells were disrupted by sonication (30 s
ON, 30 s OFF, 60%, 6 min) and the supernatant containing the
protein of interest was separated from the cell lysate by
centrifugation (10.000 rcf, 10 min, 4°C, three times). The
supernatant was incubated with 2 ml of Ni-NTA superflow
(resin, Qiagen) and 5 ml of lysis buffer [300 mM NaCl, 50 mM
NaH2PO4 pH 8.0, 20 mM imidazole, 5 mM b-mercaptoethanol,
2 mM phenylmethanesulphonylfluoride (PMSF)] for 1 h on ice
before being loaded to the column. The column was washed
with wash buffer and Spo0A was eluted in elution buffer
(300 mM NaCl, 50 mM NaH2PO4 pH 8.0, 50–250 mM imidazole) with increasing concentrations of imidazole. All protein
isolation steps were performed at 4°C. Elution fractions were
loaded on a SDS-PAGE gel to check for presence and purity of
Spo0A. Spo0A concentrations were determined with a Bradford assay using bovine serum albumin (BSA) as a standard.
DnaA was purified as previously described (Scholefield et al.,
2012).
Strains MB003, MB004, MB009 and MB010 were grown in
10 ml of LB containing kanamycin (10 mg ml-1) at 37°C and
200 r.p.m. using 100 ml bottles (Schott). The cultures were
diluted to OD600 = 0.05 in 40 ml of Schaeffer’s sporulation
medium containing 5 mg ml-1 kanamycin and kinA was
induced with 1 mM IPTG. Cultures were grown at 37°C and
225 r.p.m. using 250 ml conical shake flasks. Ten millilitres of
samples were taken between OD600 of 0.3 and 0.4 and centrifuged for 10 min at 1677 g. Samples were flash frozen in
liquid nitrogen and stored at -80°C. Samples were thawed
and dissolved in 400 ml of Diethyl-pyrocarbonate (DEPC)treated TE buffer (10 mM TRIS-HCL 1 mM EDTA, pH 7.5).
Samples were added to a 2 ml screw-cap tube on ice containing 500 mg of glass beads (75–150 mm), 175 ml of
Macaloid, 50 ml of 10% SDS and 500 ml of phenol/chloroform
mixture (1:1). Samples were placed in a Biospec
Minibeadbeater-8 and beaten 2¥ 1 min with a 1 min interval
on ice. Samples were centrifuged at 10 600 g for 10 min at
4°C. The upper phase was added to 500 ml of chloroform and
centrifuged for 5 min at 10 600 g, 4°C. Further purification
was with the Roche High Pure RNA isolation kit using 1 ml of
binding/lysis buffer. After purification, a second 1 h DNase
incubation step was performed with DNase from the Roche
kit, and the sample was purified a second time. For cDNA
preparation, 2 ml of random nonamers (1.6 mg ml-1) were
annealed to 10 mg of total RNA in a total volume of 18 ml for
5 min at 70°C. Next, 12 ml of mastermix [6 ml of 5¥ First
© 2013 Blackwell Publishing Ltd, Molecular Microbiology, 87, 925–938
936 M. Boonstra et al. 䊏
Strand buffer, 3 ml of 0.1 M DTT, 25¥ dNTP mix (12.5 mM
each dNTP), 1.8 ml of Superscript III reverse transcriptase]
was added and samples were incubated for 16 h at 42°C. To
degrade RNA, samples were treated with 3.5 ml of 2.5 M
NaOH for 15 min at 37°C. Fifteen microlitres of 2 M HEPES
free acid was added and samples were purified using a
Nucleospin Gel and PCR clean up kit. RT-QPCR was performed on a Bio-Rad iQ5 PCR machine using Bio-Rad iQ
supermix. QPCR was performed comparing MB003 with
MB004 and MB009 and MB010 dnaA transcription levels.
The housekeeping gene gyrA was used as a reference gene.
Samples were diluted to 20 ng ml-1. Efficiency of the primers
prMB022, prMB023, prMB024, prMB025 was experimentally
determined to lie between 1.95 and 2.05. The relative gene
expression was calculated using the Livak (2-DDCt) method
(Livak and Schmittgen, 2001).
Microscopy
oriC-mCherry foci (Fig. 5). Pre-cultures were inoculated from
-80°C stocks and grown overnight at 37°C in 10 ml of LB
medium at 225 r.p.m. using blue capped 100 ml bottles
(Schott-Duran) allowing for good aeration. The following
morning, the cultures were diluted to OD600 = 0.05 in 10 ml of
Schaeffer medium (Schaeffer et al., 1965) and grown for 9 h at
37°C at a speed of 250 r.p.m. using 100 ml bottles (SchottDuran). For better visualization of the oriC-mCherry foci,
0.05% xylose was added to IDJ077, IDJ078, IDJ081 and
IDJ082 cultures at mid-exponential phase. Samples for microscopy were taken 8.5 or 9 h after dilution. Two hundred
microlitres of cells were concentrated by centrifugation with a
common Eppendorf table centrifuge (5000 rcf, 1 min) and
were resuspended in 20 ml of Spizizen salts (1% ammonium
sulphate, 8% di-potassium hydrogen orthophosphate, 3%
potassium dihydrogen orthophosphate, 0.5% tri-sodium
citrate, 0.1% magnesium sulphate) before they were spotted
on a Spizizen salts agarose pad containing 1% agarose
(Sigma). It should be noted that this centrifugation step did not
alter the localization pattern of the foci or in any way changed
the origin counts, but helped in reducing background fluorescence and concentrating the number of cells per field of view
(Fig. S2). Images were taken on a Nikon Ti microscope or on
a Personal DV (Applied Precision, GEhealthcare). Nikon:
100¥ objective with a Nikon perfect focus system using the
following filters: mCherry: ex 560/55, DM 590, em 630/60. The
typical mCherry exposure time was 400 ms using Nikon’s
Intensilight as light source and a Nikon DS-QiMc camera.
Personal DV: IX71 Microscope (Olympus), CoolSNAP HQ2
camera (Princeton Instruments), 300W Xenon Light Source,
60¥ phase-contrast objective (1.25 NA), mCherry filterset
(Chroma, excitation at 572/35 nm, emission 632/60 nm);
snapshots were taken using 10% APLLC White LED light and
0.05 s exposure for phase-contrast pictures, 100% Xenon
light and typically up to 2 s exposure for mCherry detection.
Raw data were stored using softWoRx 3.6.0 (Applied Precision) and adjusted for publication using ImageJ (http://
rsbweb.nih.gov/ij/) and CorelDRAW X3 (Corel Corporation).
Adaptations for oriC-mCherry foci in KinA overproduction
strains (Figs 6 and 7). Overnight cultures were grown in 10 ml
of LB containing kanamycin (10 mg ml-1), erythromycin (0.5 mg
ml-1) and lincomycin (12.5 mg ml-1) at 37°C and 200 r.p.m.
using 100 ml bottles (Schott-Duran) and we made sure that
the OD600 of the overnight cultures did not exceed 3.5. Next,
the cultures were diluted to OD600 = 0.05 in Schaeffer’s sporulation medium containing 5 mg ml-1 kanamycin. Cultures
(10 ml in 100 ml bottles) for exponential sampling were
induced with 1 mM IPTG upon inoculation in Schaeffer’s
sporulation medium for approximately 3 h. Samples for microscopy were taken during early exponential growth at OD600
0.3–0.4 and washed with 1¥ Spizizen salts. Cultures to
be harvested during sporulation were inoculated at midexponential phase with 1 mM IPTG to induce kinA expression
and with 0.5% xylose 30 min before harvesting to produce
TetR-mCherry (total of approximately 4 h of induction).
Samples for microscopy were taken when phase bright
endospores became visible. The samples were washed with
1¥ Spizizen salts and spotted on a Spizizen salts slide containing 1% agarose to be examined by fluorescence microscopy as described above.
Adaptations for oriC-mCherry foci in Dspo0A strains
(Fig. 8). Overnight cultures were grown in 10 ml of LB containing kanamycin (10 mg ml-1) at 37°C and 200 r.p.m. using
100 ml bottles (Schott). The cultures were diluted to
OD600 = 0.05 in Schaeffer’s sporulation medium containing
5 mg ml-1 kanamycin and kinA was induced with 1 mM IPTG.
Samples for microscopy were taken at OD600 = 0.6–0.7 and
microscopy was performed as described above.
Acknowledgements
We thank Masaya Fujita for providing the Spo0A-expression
plasmids and we thank Wilfried Meijer for valuable discussions
at an early stage of this work. I.G.d.J. and O.P.K. were supported by BaCell-SysMO and the Netherlands Organisation
for Scientific Research, Earth and Life Sciences (NWO-ALW).
M.B. and O.P.K. were supported by a grant of NWO-STW in
the scope of a Simon Stevin Meester award to O.P.K. Work in
the lab of O.P.K. is carried out within the research programme
of the Kluyver Centre for Genomics of Industrial Fermentation
which is part of the Netherlands Genomics Initiative/NWO.
G.S. was supported by a BBSRC studentship and H.M. by a
Royal Society University Research Fellowship. Work in the lab
of J.-W.V. is supported by a VENI fellowship from NWO-ALW
and by a Sysmo2 grant (NWO-ALW/BBSRC/BMBF).
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Supporting information
Additional supporting information may be found in the online
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