Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA
Boonstra, Mirjam
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2017
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Boonstra, M. (2017). Bacillus subtilis: sporulation, competence and the ability to take up fluorescently labelled DNA. Rijksuniversiteit Groningen.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Chapter 2
Spo0A regulates chromosome
copy number during
sporulation by directly binding
to the origin of replication in B.
subtilis
Mirjam Boonstra*, Imke G. de Jong*, Graham Scholefield, Heath Murray, Oscar P. Kuipers and Jan-Willem Veening
Molecular Microbiology. DOI: 10.1111/mmi.12141 *These authors contributed equally.
Abstract
When starved, Bacillus subtilis cells can enter the developmental program 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 coordination 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.
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 coordination between DNA replication and sporulation results in correct chromosome copy number, and is a requirement for efficient sporulation (Murray and Errington 2008; Veening et al. 2009; Eldar 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; Wagner et al. 2009; Rahn-Lee et al. 2011). 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, 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 how wild type chromosome copy numbers and wild type fitness. Hence, additional factors are likely at play to coordinate 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 (Figure 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 DnaA-dependent DNA duplex unwinding in
vitro (Fig. 2; Castilla-Llorente 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 Spo0A-dependent 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.
Fig. 1: Relation between DNA replication and sporulation. During Sporulation, one copy of the chromosome is pumped into the forespore and one copy remains in the mother cell. It should be noted that during rapid growth, B.
subtilis keeps at least two copies
Fig. 2: (A) Schematic representation of the origin of replication (oriC) of Bacillus subtilis showing approximate positions of Spo0A- and Dnboxes (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'- TGTNATAA-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.
(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 to 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’-TGTCGAA-3’; Fig. 2A, blue) that partially overlap with functional DnaA binding sites (inverted consensus 5’-TGTGNATAA-3’; Figure 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 2C). 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 corresponding 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 wild type, 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; Muchova et al. 2004). It has been reported that Spo0A purified from E. 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 DNA-binding 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 [γ-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 (Fig. 2 and 4A).
Fig.3. DnaA binds with similar efficiency to oriCmut compared with oriCnat in vitro. The incAB PCR product (474bp, 36nM) was incubated with twofold increasing concentrations, ranging from 94 nM to 12μM, of DnaA-ADP (A) or DnaA-ATP (B). Nucleoprotein complexes were seperated on a 1.5% agarose gel and visualised by staining with ethidium bromide.
As a negative control, we used the srfAA promoter and as a positive control the abrB promoter (Molle et al. 2003). As shown in Figure 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 Figures 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.
Fig.4: Spo0A binds less efficient to
oriCmut compared to 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 Spo0A-boxes (oriCmut) compared to 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 Spo0~P does not bind. As a positive control we used the promoter region of abrB (Pabrb) to
which Spo0A~binds with high affinity (Molle et al., 2003)
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 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,
Δsda, ΔsirA or Δsda/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 forespore (Fig.5B-F). Due to strong auto-fluorescence of the phase-bright forespore compartment, it was not possible to detect oriC mCherry foci in forespores. If replication and sporulation are coordinated correctly, mother cells (as well as forespores) are expected to contain exactly one chromosome (Fig.1). As shown in Figure 5B and 5D, wild type mother cells (IDJ075, IDJ076) as well as ΔsirA 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 5F). Moreover, the fraction of Δsda mother cells containing more than one fluorescent foci was slightly elevated when it also contained the oriCmut region (strain IDJ078; average of 1.16 vs 1.18 foci; n >450) (Fig. 5C and 5F). 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 coordination between DNA replication and sporulation is not significantly affected in oriCmut cells when tested in a wild type, or in a single Δsda or ΔsirA mutant background. However, the coordination between DNA replication and sporulation was significantly affected in cells containing oriCmut when this was tested in a Δsda/ΔsirA double-mutant 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 Fig. 5F). These results demonstrate that the presence of bona fide Spo0A binding sites in the oriC region contributes to proper coordination 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 coordination between replication and sporulation since no significant defect in this coordination 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 et al. 2005).
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. (A) Example of cells with different numbers of oriC-mCherry foci representing variations in replication initiation events (arrows). B-E. Number of oriC-mCherry foici in mother cells of cultures grown in Schaeffer medium (number of cells in %) (B). Wild type (IDJ075, oriCnat), IDJ076, oriCmut), (C) Δsda (IDJ077, oriCnat IDJ078, oriCmut), (D) ΔsirA (IDJ079, oriCnat, IDJ080, oriCmut) (E) sda/sirA double mutant (IDJ081, oriCnat IDJ08, oriCmut). (F) Comparison of the average numbers of mCherry foci per cell counted for wild type (IDJ075, IDJ076), Δsda (IDJ077, IDJ078), ΔsirA (IDJ079, IDJ0480) and sda/sirA double mutant mothercells (IDJ081, IDJ082) with oriCnat and oriCmut backgrounds respectively. Significant differences (P<0.001, according to a Mann-Whitney U-test, are marked with an asterisk.
The kinA gene was placed under control of the IPTG (isopropyl-β-D-thiogalactopyranoside)-inducible promoter Pspank, and the resulting construct was introduced into the oriCnat and oriCmut TetR-mCherry strains. In addition, Pspank-kinA in the oriCmut and oriCnat background was combined with single or double mutation in sda and sirA.
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 inductions (from the start of inoculation).
A. Wild type (MB003, Pspank-kinA, oriCnat, MB004, Pspank-kinA, oriCmut).
B. Δsda (MB005, Pspank-kinA, oriCnat, MB006, Pspank-kinA, oriCmut).
C. ΔsirA (MB007, Pspank-kinA, oriCnat, MB008, Pspank-kinA, oriCmut).
D. Δsda/ΔsirA (MB009, Pspank-kinA, oriCnat, MB0010, 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.
The resulting strains, MB003-MB010, were grown in Schaeffer’s sporulation medium containing 1 mM of 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 (Fig. 6, Fig.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 ΔsirA mutant background (on average 1.07 vs 1.45 oriC foci per mother cell in
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 at mid-exponential with 1 mM IPTG and harvested for fluorescence microscopy analysis in stationary phase after 4hrs of induction when the first phase bright spores became visible).
A. Wild type (MB003, Pspank-kinA, oriCnat, MB004, Pspank-kinA, oriCmut).
B. Δsda (MB005, Pspank-kinA, oriCnat, MB006, Pspank-kinA, oriCmut).
C. ΔsirA (MB007, Pspank-kinA, oriCnat, MB008, Pspank-kinA, oriCmut).
D. Δsda/ΔsirA (MB009, Pspank-kinA, oriCnat, MB0010, 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.
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 1mM 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 DNA replication initiation and hence proper coordination with sporulation by binding to its cognate binding sites in the oriC region.
Table 1. The fraction of cells with abnormal chromosome copy numbers increases in the absence of specific Spo0A binding sites at oriC.
Discussion
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; Wagner et al. 2009, Rahn-Lee et al. 2011). 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).
Fig. 8. Induction of sporulation by Pspank-kinA in a ΔSpo0A 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 Δspo0A, Pspank-kinA oriCnat (MB016) and Δspo0A, 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.
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 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-(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 Φ29 in vivo, thereby preventing amplification of Φ29 DNA after infection which forces the phage genome to remain dormant in cells with active Spo0A (Castilla-Llorente et al. 2006). In Φ29, binding of Spo0A~P prevents formation of the replication initiation nucleoprotein complex by preventing and displacing the replication initiator protein p6 from the origins (Castilla-Llorente 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 to the native oriCnat in vitro, while DnaA still binds at near wild type efficiency (Fig.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 coordination 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 Δsda/ΔsirA 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 Δsda, but not ΔsirA single mutant cells indicates that Sda is the most important locus for coordinating replication and sporulation under these conditions. Strikingly, the role of Spo0A~P in properly coordinating 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 (Fig. 6, Fig.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 oriC 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. 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 alpha 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 (Fig. 6, Fig. 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 coordinate DNA replication with sporulation (Figure 7D). This suggests that additional, yet unknown factors contribute to the coordination 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 Table 2, 3 and 4, respectively. Bacillus subtilis strains were grown in TY or Schaeffer medium at 37°C (see below). E. coli DH5α 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 μg ml-1 of chloramphenicol, 5 μg ml-1 of kanamycin, 100 μg ml-1 of spectinomycin, 6 μg ml-1 of tetracycline, 0.5 μg ml-1 erythromycin + 12.5 μg ml-1 lincomycin (MLS) for B. subtilis and 100 μg ml-1 of ampicillin or 150 μg ml-1 of erythromycin for E. coli. 1.5 % agar 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 PCR’s were performed with Phusion (NEB, UK), unless stated otherwise. Oligonucleotides were purchased from Biolegio (Nijmegen, NL). 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. 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 (Guerout-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.
Spo0A and DnaA purification
E. 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 DNA-binding 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 sec ON, 30 sec 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, 3 times). The supernatant was incubated with 2 ml Ni-NTA superflow (resin, Qiagen) and 5 ml lysis buffer (452 300 mM NaCl, 50 mM NaH2PO4 pH 8.0, 20 mM imidazole, 5 mM β-mercaptoethanol, 2 mM phenylmethanesulfonylfluoride (PMSF)) for one hour 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).
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 2.
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 [γ-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 BSA. 15 [l of the mix was loaded onto a native polyacrylamide gel (4 % (v/v) polyacrylamide, 1 x TBE buffer, 0.125 % (w/v) APS, 0.0125 % (v/v) TEMED) and electrophoresed in 1x 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 two-fold steps from 12 μm 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
Strains MB003, MB004, MB009 and MB010 were grown in 10 ml LB containing kanamycin (10μg/ml) at 37°C and 200rpm using 100 ml bottles (Schott). The cultures were diluted to OD600 = 0.05 in 40 ml Schaeffer’s sporulation medium containing 5 μg/ml kanamycin and kinA was induced with 1mM IPTG. Cultures were grown at 37°C and 225rpm using 250ml conical shake flasks. 10ml samples were taken between OD600 = 0.3-0.4 and centrifuged for 10min at 1677 g. Samples were flash frozen in liquid nitrogen and stored at -80 °C. Samples were thawed and dissolved in 400μl Diethyl-pyrocarbonate (DEPC) treated TE buffer (10mM TRIS-HCL 1mM EDTA, pH 7.5).
Samples were added to a 2ml screw-cap tube on ice containing 500mg glass beads (75-150 μm), 175μl Macaloid, 50μl 10% SDS and 500μl phenol/chloroform mixture (1:1). Samples were placed in a Biospec Minibeadbeater- 8 and beaten 2X 1 min with a 1 min interval on ice. Samples were centrifuged at 10600g for 10min at 4°C. The upper phase was added to 500μl chloroform and centrifuged for 5min at 10600g, 4°C. Further purification was with the Roche High Pure RNA isolation kit using 1ml binding/lysis buffer. After purification, a second 1hr DNase incubation step was performed with DNase from the Roche kit, and the sample was purified a second time. For cDNA preparation, 2μl random nonamers (1.6μg/ml) were annealed to 10μg of total RNA in a total volume of 18μl for 5min at 70°C. Next, 12μl mastermix (6μl 5X First Strand buffer, 3μl 0.1M DTT, 25X dNTP mix (12.5mM each dNTP), 1.8μl Superscript III reverse transcriptase) was added and samples were incubated for 16hrs at 42°C. To degrade RNA, samples were treated with 3.5 μl 2.5M NaOH for 15min at 37°C. 15μl 2M HEPES free acid was added and samples were purified using a Nucleospin Gel and PCR clean up kit. RT-QPCR was done on a Biorad iQ5 PCR machine using Biorad 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 20ng/μl. 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-ΔΔCt516 ) method (Livak and Schmittgen 2001).
Microscopy
oriC-mCherry foci (Figure 5): Precultures were inoculated from -80°C
stocks and grown overnight at 37°C in 10 ml LB medium at 225 rpm using blue capped 100ml bottles (Schott-Duran) allowing for good aeration. The following morning, the cultures were diluted to OD600 = 0.05 in 10 ml Schaeffer medium (Schaeffer et al. 1965) and grown for 9 hours at 37°C at a speed of 250 rpm using 100 ml bottles (Schott-Duran). 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. 200 μl cells were concentrated by centrifugation with a common Eppendorf table centrifuge (5000 rcf, 1 min) and were resuspended in 20 μl 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: 100X 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 400ms 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, 60x 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 (Figures 6 and 7): Overnight cultures were grown in 10 ml LB containing kanamycin (10 [g/ml), erythromycin (0.5 [g/ml) and lincomycin (12.5 [g/ml) at 37°C and 200 rpm 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 [g/ml of kanamycin. Cultures (10 ml in 100 ml bottles) for exponential sampling were induced with 1 mM of IPTG upon inoculation in Schaeffer’s sporulation medium for approximately 3h. Samples for microscopy were taken during early exponential growth at OD600 0.3-0.4 and washed with 1x Spizizen salts.
Cultures to be harvested during sporulation were inoculated at mid exponential 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 4h of induction). Samples for microscopy were taken when phase bright endospores became visible. The samples were washed with 1x 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 Δspo0A strains (Figure 8): Overnight cultures were grown in 10 ml LB containing kanamycin (10 [g/ml) at 37°C and 200rpm using 100 ml bottles (Schott). The cultures were diluted to OD600 = 0.05 in Schaeffer’s sporulation medium containing 5 [g/ml kanamycin and kinA was induced with 1mM 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. IDJ and OPK were supported by BaCell-SysMO and the Netherlands Organisation for Scientific Research, Earth and Life Sciences (NWO-ALW). MB and OPK were supported by a grant of NWO-STW in the scope of a Simon Stevin Meester award to OPK.
Work in the lab of OPK 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. GS was supported by a BBSRC studentship and HM by a Royal Society University Research Fellowship. Work in the lab of JWV is supported by a VENI fellowship from NWO-ALW and by a Sysmo2 Grant (NWO581 ALW/BBSRC/BMBF). Supporting information
Additional supporting information may be found in the online version of this article.
References
Burbulys D, Trach KA, and Hoch JA (1991) Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell, 64, 545-552.
Burkholder WF, Kurtser I, and Grossman AD (2001) Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis. Cell, 104, 269-279.
Castilla-Llorente V, Munoz-Espin D, Villar L, Salas M, and Meijer WJ (2006) Spo0A, the key transcriptional regulator for entrance into sporulation, is an inhibitor of DNA replication. The EMBO Journal, 25, 3890-3899.
Collier J (2012) Regulation of chromosomal replication in Caulobacter crescentus. Plasmid. 67(2):76-87.
Cunningham KA and Burkholder WF (2009) The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F. Mol Microbiol, 71, 659-677.
Eldar A, Chary VK, Xenopoulos P, Fontes ME, Loson OC, Dworkin J, Piggot PJ, and Elowitz MB (2009) Partial penetrance facilitates developmental evolution in bacteria. Nature, 460, 510-514.
Errington J (2001) Septation and chromosome segregation during sporulation in Bacillus subtilis. Curr Opin Microbiol, 4, 660-666.
Errington J (2003) Regulation of endospore formation in Bacillus subtilis. Nat Rev Micro, 1, 117-126.
Fujita M and Losick R (2003) The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division. Genes Dev, 17, 1166-1174.
Fujita M and Losick R (2005) Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A. Genes Dev, 19, 2236-2244.
Fukuoka T, Moriya S, Yoshikawa H, and Ogasawara N (1990) Purification and characterization of an initiation protein for chromosomal replication, DnaA, in Bacillus subtilis. J Biochem, 107, 732-739.
Guérout-Fleury AM, Frandson N, and Stragier P (1996) Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 57-61
Harwood CR and Cutting SM (1990). Molecular biological methods for Bacillus. John Wiley and sons Ltd., Chichester.
Higgins D, and Dworkin J (2012). Recent progress in Bacillus subtilis sporulation. FEMS Microbiol Rev. 36(1):131-48.
Ireton K, Rudner DZ, Siranosian KJ, Grossman AD (1993) Integration of multiple developmental signals in Bacillus subtilis through the Spo0A transcription factor. Genes Dev. 7(2):283-294.
Itaya M (1992) Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome Biosc Biotech Biochem, 56, 685-686.
de Jong IG, Veening JW, and Kuipers OP (2010) Heterochronic phosphorelay gene expression as a source of heterogeneity in Bacillus subtilis spore formation. J Bacteriol, 192, 2053-2067.
Kolter R (2010) Biofilms in lab and nature: a molecular geneticist's voyage to microbial ecology. Int Microbiol, 13, 1-7.
Krause M, Rückert B, Lurz R, and Messer W (1997) Complexes at the replication origin of Bacillus subtilis with homologous and heterologous DnaA protein. J Mol Biol, 274, 365-80. Ladds JC, Muchová K, Blaskovic D, Lewis RJ, Brannigan JA, Wilkinson AJ, and Barák I (2003) The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli. FEMS Microbiol Lett, 223, 153-157.
Lewis RJ, Krzywda S, Brannigan JA, Turkenburg JP, Muchová K, Dodson EJ, Barák I, Wilkinson AJ (2000) The trans-activation domain of the sporulation response regulator Spo0A revealed by X-ray crystallography. Mol Microbiol. 38(2):198-212.
Lewis RJ, Scott DJ, Brannigan JA, Ladds JC, Cervin MA, Spiegelman GB, Hoggett JG, Barák I, and Wilkinson AJ (2002) Dimer formation and transcription activation in the sporulation response regulator Spo0A. J Mol Biol, 316, 235-245.
Livak KJ, and Schmittgen TD (2001) Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods, 25, 402-408.
Lopez D and Kolter R (2010): Extracellular signals that define distinct and coexisting cell fates in Bacillus subtilis. FEMS Micriobiol Rev, 34, 134-149.
Messer W (2002) The bacterial replication initiator DnaA. DnaA and oriC, the bacterial mode to initiate DNA replication. FEMS Microbiol Rev, 26, 355-374.
Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, and Losick R (2003) The Spo0A regulon of Bacillus subtilis. Molecular Microbiology, 50, 1683-1701. Moriya S, Fukuoka T, Ogasawara N, Yoshikawa H (1988) Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the Bacillus subtilis chromosome. EMBO J. 7(9):2911-2917.
Moriya S, Imai Y, Hassan A, and Ogasawara N (1999) Regulation of initiation of Bacillus subtilis chromosome replication. Plasmid, 41, 17-29.
Muchová K, Lewis RJ, Perecko D, Brannigan JA, Ladds JC, Leech A, Wilkinson AJ, and Barák I (2004) Dimer-induced signal propagation in Spo0A. Mol Microbiol. 53(3):829-842.
Murray H and Errington J (2008) Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. Cell, 135, 74-84.
Ogasawara N, Moriya S, Yoshikawa H (1985) Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. IV. Transcription of the oriC region and expression of DNA gyrase genes and other open reading frames. Nucleic Acids Res.13(7):2267-2279.
Rahn-Lee L, Gorbatyuk B, Skovgaard O, and Losick R (2009) The conserved sporulation protein YneE inhibits DNA replication in Bacillus subtilis. J Bacteriol, 191, 3736-3739. Rahn-Lee L, Merrikh H, Grossman AD, and Losick R (2011) The sporulation protein SirA inhibits the binding of DnaA to the origin of replication by contacting a patch of clustered amino acids. J Bacteriol, 193, 1302-1307.
Rowland SL, Burkholder WF, Cunningham KA, Maciejewski MW, Grossman AD, and King GF (2004) Structure and mechanism of action of Sda, an inhibitor of the histidine kinases that regulate initiation of sporulation in Bacillus subtilis. Mol Cell, 13, 689-701.
Ruvolo MV, Mach KE, and Burkholder WF (2006) Proteolysis of the replication checkpoint protein Sda is necessary for the efficient initiation of sporulation after transient replication stress in Bacillus subtilis. Mol Microbiol, 60, 1490-508.
Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Sekimizu K, Bramhill D, and Kornberg A (1987) ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell, 50(2):259-265. Schaeffer P, Millet J, and Aubert J-P (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci USA, 54, 704-711.
Scholefield G, Errington J, Murray H (2012) Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA. EMBO J. 31(6):1542-1555.
Strauch M, Webb V, Spiegelman G and Hoch JA (1990) The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene. Proc Natl Acad Sci USA, 87, 1801-1805.
Veening JW, Murray H, and Errington J (2009) A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis. Genes Dev, 23, 1959-1970.
Wang JD, Sanders GM, and Grossman AD (2007) Nutritional control of elongation of DNA replication by (p)ppGpp. Cell, 128, 865-75.
Wagner JK, Marquis KA, and Rudner DZ (2009) SirA enforces diploidy by inhibiting the replication initiator DnaA during spore formation in Bacillus subtilis. Mol Microbiol, 73, 963-974.
Whitten A, Jacques D, Hammouda B, Hanley T, King GF, Guss JM, Trewhella J, and Langley DB (2007) The structure of the KinA-Sda complex suggests an allosteric mechanism of histidine kinase inhibition. J Mol Biol, 368, 407-420.
Xenopoulos P and Piggot PJ (2011) Regulation of growth of the mother cell and chromosome replication during sporulation of Bacillus subtilis. J Bacteriol, 193, 3117-26.
