Dual-Specificity Anti-sigma Factor Reinforces Control of Cell-Type Specific Gene Expression in Bacillus subtilis

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.


Introduction
Developmental transcription networks underlie all cellular differentiation processes. These networks usually integrate a variety of environmental and cellular inputs to activate regulators such as transcription factors that control the expression of cell type-specific genes [1,2]. Superimposed onto these transcription networks, are protein-protein interaction, signal transduction and metabolic networks [3]. Overall, the combination of these different networks ensures the timely production of proteins and other components essential for the morphogenesis of newborn cells. Ultimately, a complete understanding of cellular differentiation requires detailed knowledge of how the circuitry of transcriptional regulators influences global gene expression in space and time to drive cell morphogenesis at sequential stages of development.
Spore formation in the bacterium Bacillus subtilis is an example of a prokaryotic cell differentiation process. At the onset of sporulation, triggered by severe nutrient scarcity, the rodshaped cell divides close to one of its poles producing a small forespore, the future spore, and a larger mother cell (Fig. 1A). The mother cell nurtures development of the forespore, but undergoes autolysis to release the mature spore at the end of the process. Soon after asymmetric division, the mother cell engulfs the forespore, which becomes isolated from the external medium and separated from the mother cell cytoplasm by a double membrane and an intermembrane space. Following engulfment completion, gene expression in the mother cell drives the last stages of spore maturation by promoting the assembly of concentric protective structures. In parallel, gene expression in the forespore prepares the future spore for dormancy.
The sporulation regulatory network includes four RNA polymerase sigma subunits that are activated in a cell type-specific manner and define a regulatory cascade that constitutes the core of the transcription network. σ F and σ E control early stages in development in the forespore and in the mother cell, respectively. At late stages of development, i.e., post-engulfment, σ F is replaced by σ G , and σ E is replaced by σ K (Fig. 1A). The genes for the cell type-specific sigma factors are part of a genomic signature for sporulation [4,5,6,7]. A second level of regulation results from the action of additional transcription factors, three in the mother cell (SpoIIID, GerR, and GerE), and two in the forespore (RsfA and SpoVT), which are less conserved among spore-formers. The combination of the primary regulators (i.e., the sporulation sigma factors) and the secondary regulators (ancillary transcription factors) organizes the two cell-specific lines of gene expression into a series of interlocked type-1 coherent and incoherent feed-forward loops (FFLs) [8,9] (Fig. 1B). Coherent type-1 FFLs are used as persistence detectors whereas incoherent type-1 FFL generate pulses of gene expression (reviewed by [3]).
Superimposed onto the transcriptional network are several cell-cell signaling pathways that operate at critical stages of morphogenesis, across the forespore membranes, to allow activation of the next sigma factor in the cascade, in the adjacent cell (Fig. 1B). The requirement for σ G for σ K activity is an example of such a pathway. σ F drives the initial transcription of the gene for σ G in the forespore [13], but the main period of σ G activity, dependent on activation of an auto-regulatory loop, only begins after engulfment completion [14,15,16,17]. σ G then controls production of a signaling protein, SpoIVB, which is secreted into the intermembrane space and activates the machinery responsible for the proteolytical activation of σ K [18].
Another level of control of sigma factor activity is through the inhibitory action of antisigma factors. CsfB (also called Gin, for inhibitor of sigma G) is a Zn 2+ -containing anti-sigma factor that inhibits σ G [19,20,21,22]. CsfB is part of a composite negative feedback loop that together with another anti-sigma factor, SpoIIAB, and the LonA protease, prevents activation of the auto-regulatory σ G in pre-divisional cells (Fig. 1C) [20]. After the onset of sporulation CsfB is also produced in the forespore under σ F control [23], and has a role in delaying the onset of σ G activity until engulfment completion [14,24]. Importantly, while σ F and σ G are highly similar proteins, σ F itself is refractory to the action of CsfB. The basis for this selectivity can be traced to a single asparagine residue in a conserved β´-interacting surface of σ G [20]. Likewise, a conserved glutamate residue of σ F is important for resistance (Fig. 1D). The anti-σ G activity of CsfB, and the delayed transcription of the gene encoding σ G relative to other σ F -dependent genes are redundant mechanisms ensuring that the onset of σ G activity begins at the appropriate time, possibly to coincide with engulfment completion. Accurate temporal control of σ G activity is important, because it leads to the activation of σ K [18] and premature activity of σ K interferes with spore morphogenesis [25]. Timely activation of σ K is important, as it ensures that the final stages in the assembly of the spore surface structures only initiate after the forespore is engulfed [18]. The σ G -dependent production of SpoIVB, together with delayed transcription of genes required for pro-σ K synthesis and processing, which require both σ E and SpoIIID ( Fig. 1B; above), effectively couples pro-σ K activation to engulfment completion. In total, timely activation of the cell type-specific sigma factors enforces the directionality and fidelity of the morphogenetic process [7,18].
This works focuses on an additional role of the anti-sigma factor CsfB in controlling cell type-specific gene expression during spore development. We show that in addition to its previously characterized functions in the forespore and pre-divisional cells, the anti-sigma factor CsfB also plays a role in the mother cell where its synthesis is activated under the control of σ K . We show that CsfB binds to and inhibits σ E in vitro and in vivo, while σ K is resistant to CsfB. A single residue in conserved region 2.3 of the sigma subunit is sufficient for the discrimination by CsfB and swapping this specificity interferes with the temporal progression of the mother cell line of gene expression. Thus, CsfB is part of a negative feedback loop that acts to limit the activity of σ E following engulfment completion. Furthermore, we show that CsfB is also involved in preventing ectopic activity of σ G in the mother cell. Therefore, CsfB is intricately connected to both cell-specific lines of gene expression to enforce the cell-type specific action of σ G and the early to late developmental transition in the two cell types undergoing sporulation.

Results
Following engulfment completion, expression of csfB is turned on in the mother cell from a σ K -dependent promoter controlled promoters [8] (Fig. 2A). Presumably, the same σ F -type promoter can also be recognized by σ G in pre-divisional cells [8,20].
In addition, sequences matching the -10 (CATATACT) and -35 (AACACCGA) elements of the σ K consensus binding sequence are present in the csfB regulatory region, upstream of the putative σ F promoter [26] (Fig. 2A). This suggested to us that expression of csfB could also take place in the mother cell, at a later stage in development, after σ K is activated [25]. To test this possibility, we first examined expression of a functional csfB-gfp fusion inserted at the non-essential amyE locus of a strain deleted for the native csfB gene [20]. Cells were induced to sporulate by resuspension in a nutrient poor medium and examined by phase contrast and fluorescence microscopy at different times after resuspension (which marks the beginning of sporulation under these culturing conditions). In a wild type background, expression of csfB-gfp was first detected in the Expression of csfB in sporulating cells. A: the panel represents the regulatory region of the csfB gene, with the -35 and -10 elements of putative σ For σ K -dependent promoters. The consensus sequences for promoter recognition by σ F or σ K are represented below the promoter sequence (R is A or G; W is A or T; Y is C or T; N, is any base) [8,9]. The point mutations introduced in the putative σ F -or σ K -dependent promoters are indicated in red. The ribosomebinding site (RBS) and start codons are also represented, for reference. B and C: the localization of a CsfB-GFP fusion protein, expressed from the amyE locus in a csfB mutant background was monitored by fluorescence microscopy at the indicated times (in hours) after the onset of sporulation in re-suspension medium, in cultures of the following strains: an otherwise wild type, a ΔsigK mutant, a strain expressing CsfB-GFP from the forespore-specific P spoIIQ promoter (in B); strains bearing the point mutations indicated in A, in the σ F (P sigK ) or σ K promoters (P sigF ) (in C). Cells were stained with the membrane dye FM4-64 prior to fluorescence microscopy. Scale bars, 1 μm. The cartoons in B represent the percentage of cells with GFP fluorescence in the coloured compartment at hour 4 of sporulation in re-suspension medium. D: in vitro transcription reactions using RNA polymerase containing the indicated B. subtilis sigma factors, and a PCR template containing the csfB promoter. Products of about 148 or 220 nucleotides were expected for transcripts initiating from the putative σ F -and σ K -dependent promoters, as represented. forespore of sporulating cells (sporangia), two hours after the onset of sporulation, consistent with the time of activation of σ F (Fig. 2B) [20]. However, by hour 3 of sporulation CsfB-GFP fluorescence was also observed in the mother cell of sporangia that had completed the process of forespore engulfment, as indicated by the loss of FM4-64 staining at the forespore membranes ( Fig. 2B). At hour 4 of sporulation, 55% of sporangia exhibited a fluorescence signal restricted to the forespore, 30% fluoresced only in the mother cell, and 15% in both cell types (Fig. 2B). Moreover, the appearance of a CsfB-GFP signal in the mother cell seemed to occur concomitantly with a decrease in the forespore-specific fluorescence signal (Fig. 2B). By contrast, no CsfB-GFP fluorescence was seen in the mother cell of a sigK deletion mutant, even though these sporangia remained capable of completing engulfment (Fig. 2B). These results suggest that σ K is responsible for the mother cell-specific accumulation of CsfB-GFP.
An alternative explanation, however, is that CsfB-GFP may be exported from the forespore to the mother cell upon engulfment completion, in an unindentified process that would require σ K activity. To test this idea we replaced the native promoter sequences (P csfB ) driving expression of the csfB-gfp fusion by P spoIIQ , a well-characterized forespore-specific, σ F -dependent promoter [22,27,28]. In this strain, accumulation of CsfB-GFP was detected in the forespore from hour 2 of sporulation onwards as expected, but was never observed in the mother cell even after σ K had been activated (Fig. 2B). Thus, mother cell-specific transcription, and not transport from the forespore, is responsible for CsfB-GFP accumulation in the mother cell at late stages of development.
To more precisely delineate the respective contributions of the csfB promoters, we introduced point mutations in each of the σ F -and σ K -type putative -10 elements (highlighted in red in Fig. 2A) and examined production of CsfB-GFP during sporulation. In a strain bearing mutations in the putative σ K -10 element, in which csfB-gfp expression is presumably only driven by the σ F -dependent promoter (P sigF in Fig. 2C), CsfB-GFP is restricted to the forespore. Conversely, when point mutations were introduced in the -10 region of the putative σ F -dependent promoter (P sigK in Fig. 2C), CsfB-GFP only accumulates in the mother cell.
Immunoblot analysis of whole cell extracts of sporulating cells producing CsfB-GFP under the control of the wild type promoters revealed a steady increase in the accumulation of the fusion protein, between hour 2 and 5 of sporulation (S1A Fig). By contrast, when expression of the fusion was solely dependent on P sigF , the intensity of the CsfB-GFP band strongly decreased after hour 3, whereas when expression was driven exclusively from P sigK , CsfB-GFP accumulated only from hour 3 onwards (S1A Fig). These observations are consistent with the presumed windows of activity of each of the csfB promoters ( Fig. 2A), as well as the fluorescence microscopy experiments described above ( Fig. 2B and C). We also carried out experiments with a lacZ transcriptional fusion and similarly detected two periods of csfB expression, the first beginning around hour 2 of sporulation and the second around hour 3-4 (S1B Fig). The first period of β-galactosidase activity was not observed when expression of the fusion relied only on P sigK , whereas the second period was absent when expression of csfB was controlled by P sigF (S1B Fig). These results are in line with the view that expression of csfB takes place at two developmental stages during sporulation, first in the forespore, under the control of σ F , and later in the mother cell, under the control of σ K .
Finally, we conducted in vitro transcription reactions in which core RNA polymerase, purified from B. subtilis, was reconstituted either with each of the four cell type-specific sporulation sigma factors or with the main sigma subunit, σ A . All σ factors were overproduced and purified from E. coli cells (S2A Fig; see also S1 Text). While the σ A or σ E forms of RNA polymerase did not initiate transcription from the csfB promoter region, a run-off product of 148 nucleotides was obtained with the σ F -and σ G -containing holoenzymes (Fig. 2D). The size of this transcript is consistent with the location of P sigF and in line with the idea that σ F and σ G utilize the same csfB promoter ( Fig. 2A). In addition, a product of 220 nucleotides was obtained with the σ Kreconstituted holoenzyme (Fig. 2D), in agreement with the location of the predicted P sigK ( Fig. 2A). In all, our findings suggest that, following engulfment completion, expression of csfB is switched off in the forespore by an unknown mechanism and activated in the mother cell, under the direct control of σ K .
CsfB modulates expression of the σ E and σ K regulons In pre-divisional sporangia, σ G activity is inhibited by a composite negative-feedback loop involving CsfB [20]. We have argued that the σ F -promoter of csfB is recognized and used by σ G to achieve this mechanism of negative auto-regulation (Fig. 1C). Thus, inactivation of P sigF should mimic the previously described activity of σ G in pre-divisional cells of a csfB deletion mutant [20]. In support of this hypothesis, we found that P sigK cells (carrying mutations in the σ F promoter) show activity of σ G in pre-divisional cells, as measured by the expression of transcriptional fusions of lacZ and cfp to the σ G -dependent sspE promoter (S3 Fig; see also S1 Text). These results confirm that the σ F -type promoter of csfB is the one used by σ G in predivisional cells.
Next, we used DNA microarrays to examine sporulation-specific gene expression in the P sigF strain, which does not express CsfB in the mother cell, in comparison to the wild type. The total RNA used for these studies was extracted and purified from cultures in resuspension medium at hour 3 of sporulation, i.e., shortly after csfB is turned on in the mother cell (above). We observed important changes in expression of several of the sporulation sigma factor regulons. Nevertheless, the expression of nearly all of the genes in the σ F regulon remained unchanged in the P sigF strain versus the wild type ( Fig. 3A; S3 Table); however, this does not include the group of forespore-expressed genes that are under the dual control of σ F and σ G (see below, σ G regulon). By contrast, a large fraction of the σ E -dependent genes was upregulated in the P sigF strain relative to the wild type ( Fig. 3A; S3 Table). Importantly, the σ E regulon can be sub-divided into 4 groups: i) genes that rely on σ E alone for expression, ii) those that require SpoIIID as a positive factor, iii) those that are repressed by SpoIIID, and iv), those that are repressed by GerR [9]. A closer inspection of the transcriptional profiling data revealed that the σ Edependent genes that are upregulated in the P sigF strain are the first and second groups, i.e., those that rely exclusively on σ E for expression and those that are activated by SpoIIID ( Fig. 3A; S3 Table). Conversely, essentially no upregulated genes are seen among the σ E -dependent genes that are repressed by GerR or SpoIIID ( Fig. 3A; S3 Table). These genes correspond to pulses X 2 and X 3 in Fig. 1B [9]. It is likely that transcription of these genes has already been turned off by the time CsfB starts to accumulate in the mother cell, explaining the lack of effect of the mutation. In contrast, the increased expression of the σ Eand σ E /SpoIIID-dependent genes suggests that in the absence of CsfB, the activity of σ E in the mother cell is increased or prolonged.
Several of the σ E /SpoIIID-dependent genes are involved in production and activation of σ K [9,29]. The σ K regulon itself can be sub-divided into genes whose expression is activated by σ K alone, those that require GerE for full expression (pulse X 7 in Fig. 1B) and those repressed by GerE (pulse X 6 ) [9]. Transcriptional profiling of the P sigF strain shows that a significant fraction of the σ K -dependent genes is upregulated, regardless of GerE dependency ( Fig. 3A; S3 Table). We presume that the increased expression of the σ K regulon may result from augmented activity of σ K (but see also below, section on the ectopic activation of the σ G regulon).
In all, the genome-wide transcriptional profiling data highlight the importance of csfB for modulating the levels of σ E -and σ K -dependent gene expression in the mother cell, and are consistent with the interpretation that perturbations affecting the mother cell line of gene expression in a csfB mutant are mainly caused by an increased and protracted activity of σ E .
CsfB contributes to the inhibition of σ G and the timely activation of σ K in the mother cell A somewhat unexpected result of the global transcriptional profiling analysis was that the mutation in the σ K -dependent promoter of csfB also caused a generalized increase in the expression of the σ G regulon ( Fig. 3A; S3 Table), including genes that are repressed (pulse X 4 in Fig. 1B) or activated by SpoVT (pulse X 5 ). We considered two possibilities. First that the increase in σ E activity in the P sigF strain somehow led to an increase in the activity of σ G in the forespore. Second, that eliminating expression of csfB in the mother cell alleviated some of the restrictions imposed on σ G to become active in this cell type. To test these hypotheses, we first examined the localization of the fluorescence signal from a P sspE -CFP fusion in cells of the P sigF -csfB strain, in comparison with the wild type, at hour 3 of sporulation in resuspension medium. In the wild type strain, and as expected for a σ G -controlled gene, the fluorescence signal Role of CsfB in the mother cell. A: microarrays were used to assess the impact of mutational inactivation of the σ G -dependent promoter for the csfB gene (P sigF strain) on sporulation-specific gene expression. The panel represents the percentage of genes in each of the sporulation-specific sigma regulons, whose expression was increased (green bars) or unaffected (grey bars) in the P sigF mutant relative to the wild type. Genes, repressed ("R") by or dependent ("D") on the following ancillary transcription factors, are shown: D, SpoIIID; R, GerR; VT, SpoVT; E, GerE. B: the panel illustrates the results of a fluorescence in situ hybridization (FISH) experiment to localize the sspE mRNA, produced under σ G control, in a wild type strain and in the P sigF mutant. The mRNA was localized using a fluorescein-labelled anti-sense oligonucleotide and fluorescence microscopy. PC, phase contrast. The graph on the right shows the length (in μm) distribution of the fluorescence signal measured along the longitudinal axis of the cells. C: immunoblot analysis of pro-σ K and σ K . Samples from cultures of the wild type and P sigF -csfB strain were collected at the indicated times (in minutes) after the induction of sporulation by re-suspension. Whole cell extracts were prepared, proteins (15 μg) electrophoretically resolved and immunobloted with anti-σ K and anti-σ A (as a loading control) antibodies. Arrows indicate the position of pro-σ K (in black) and σ K (red).
doi:10.1371/journal.pgen.1005104.g003 was confined to engulfed forespores (S4A Fig, yellow arrows). Fluorescence from P sspE -cfp was also detected in engulfed forespores for the P sigF strain (S4A Fig, yellow arrows). However, for the P sigF mutant, a week fluorescence signal was also detected in the mother cell prior to engulfment completion (S4A Fig, white arrows). The quantitative analysis of the signal shows that the mother cell-associated fluorescence in P sigF sporangia that have not completed the engulfment sequence is consistently higher than for the wild type (S4B Fig; see also S1 Text).
In an attempt to verify this observation, we turned to fluorescence in situ hybridization (FISH) using a specific antisense DNA probe labelled with Cy3, in an attempt to localize the sspE mRNA [30]. Because at this time in sporulation the forespore is not yet recognizable by phase contrast microscopy, we relied mainly on the size of the fluorescence signal to distinguish mother cell or sporangia (larger) from forespore (smaller) localization. The FISH images suggest that the signal is confined to the forespore in the wild type strain, whereas the signal is mostly associated with whole cells (sporangia) in the P sigF strain (Fig. 3B). In cells of the mutant strain, the size of the fluorescence signal is 1.5 to 2 times longer than that observed for the wild type strain (Fig. 3B). Moreover, the size of the fluorescence signal for the P sigF strain coincides with the size of the sporangia as judged from the phase contrast images (Fig. 3B). This suggests whole cell production of the sspE mRNA. Some cells of the P sigF mutant do not show a forespore-associated signal (Fig. 3B, insert). This suggests that expression of sspE may initiate earlier in the mother cell than in the forespore, in line with the detection of P sspE -CFP in the mother cell, for the P sigF strain, prior to engulfment completion (S4 Fig; see also above). In any event, the results are consistent with the hypothesis that the sspE mRNA is also produced in the mother cell. Therefore, these data suggest that eliminating transcription of csfB from the P sigK promoter increases transcription of σ G -dependent genes in the mother cell and thus that CsfB contributes to the negative regulation of σ G in this cell. Presumably, CsfB is part of a composite negative feedback loop limiting σ G activity in the mother cell similar to the mechanism that prevents activation of σ G in pre-divisional cells. In these cells, CsfB plays a key role in inhibiting σ G activity, along with the SpoIIAB anti-sigma factor and the LonA protease [20,31,32]. Previous work has shown that when the sigG gene is placed under the control of a σ E -controlled promoter, SpoIIAB and LonA are also important for the inhibition of σ G activity in the mother cell [15,20,32]. In an extension of this work, we now show that SpoIIAB plays the leading role in σ G inhibition in the mother cell, while LonA and CsfB appear to have largely redundant supportive contributions (S5A Fig). Nevertheless, these results demonstrate the ability of CsfB to act as an inhibitor of σ G activity in the mother cell, in line with the observed σ G activity in the P sigF strain ( Fig. 3 and S4 Fig).
Importantly, during the course of these experiments aiming at directly testing for a role of CsfB in the inhibition of σ G in the mother cell (above), we noticed that the forced production of σ G in the mother cell resulted in premature activity of σ K (S5B Fig). Activation of σ K requires pro-σ K processing, which in turn depends on the activity of σ G in the forespore, following engulfment completion [25,33]. Since σ G shows some activity prior to engulfment completion in the P sigF strain (above), we therefore considered the possibility that the augmented transcription of the σ K -controlled genes, as detected in our microarray analysis (above), could result in part from premature processing of pro-σ K independently of the σ G -dependent pathway that operates following engulfment completion (S5C Fig). To test this idea, we surveyed the accumulation of pro-σ K and σ K by immunoblotting, throughout sporulation. Pro-σ K was first detected in both the wild type strain and in the P sigF strain 140 min after resuspension in sporulation medium (Fig. 3C). However, mature σ K is first detected 180 min after resuspension for the P sigF strain, and only at minute 200 for the wild type (Fig. 3C). Moreover, even at minute 200, the fraction of mature σ K is higher for the P sigF strain (Fig. 3C). Therefore, the lack of csfB expression in the mother cell, can lead to premature activation of pro-σ K , again underscoring the need for strict inhibition of σ G activity in the mother cell.

CsfB forms a complex with σ E but not with σ K in sporulating cells
The analysis of the global transcriptional profiling data raised the possibility that CsfB could act as an inhibitor of σ E and/or σ K . This prompted us to determine whether the anti-sigma factor could form complexes with either sigma factor. To isolate CsfB-GFP and interacting proteins from sporulating cultures of a strain expressing csfB-gfp, we used a GFP-binding protein (GBP) coupled to a chromatographic matrix (GFP-Trap beads). As controls, we examined a wild type strain carrying no gfp fusion and a strain producing GFP under the control of the xylose-inducible P xylA promoter. The extracts were prepared 4 hours after the onset of sporulation, when CsfB-GFP is known to accumulate in the mother cell ( Fig. 2B; above). Control experiments also confirmed the accumulation of σ E , σ G , σ K or GFP in the whole-cell extracts prepared from the various strains (Fig. 4A). The extracts from all strains were incubated with GFP-Trap beads, the bound proteins eluted and identified by immunoblot analysis with antibodies raised against σ E , σ G , σ K or GFP. We found σ E but not σ K , to be pulled down efficiently from extracts of the strain producing CsfB-GFP. By contrast, σ E was not recovered from extracts of the strain containing no GFP fusion or from the strain that produced unfused GFP (Fig. 4A). Thus, retention of σ E by the GFP-trap beads depended on formation of a complex with CsfB-GFP. As expected, CsfB-GFP was able to pull down σ G [20], a property used here as a positive control for the experiment (Fig. 4A). Interestingly, a role for CsfB in inhibiting σ E activity in the forespore soon after asymmetric division was previously suggested [34]. Under the conditions of our experiments, however, σ E is only expected to accumulate in the mother cell and the expression of csfB has switched to the mother cell in most sporangia in the population when the cells were harvested for the pull down assays (see above). Therefore, the result reported in Fig. 4A most likely reflects an interaction occurring between σ E and CsfB in the mother cell and not in the forespore.
In agreement with this interpretation, CsfB-GFP produced solely from the P sigK promoter, was still able to pull down σ E (Fig. 4A). In addition, this construct could also pull down σ G and conversely CsfB-GFP produced from the P sigF was able to pull down σ E (Fig. 4A). Even though these observations could be explained by complex formation after the cells are disrupted in particular for the P sigF strain (provided that there was enough free CsfB-GFP), we note that more σ E and less σ G seemed to be pulled-down in the strain where csfB-gfp expression is restricted to the mother cell than in the strain producing CsfB-GFP in both compartments (Fig. 4A). Thus, it is possible that the amounts of σ E and σ G pulled-down from the P sigK -csfB-gfp carrying strains reflect the levels of both of these proteins in the mother cell. Considering that σ G can accumulate and become active in the mother cell under certain conditions [15,20] CsfB binds to σ E but not to σ K One of the consequences of inactivating the P sigK promoter of csfB was the protracted expression of a specific class of σ E -dependent genes. A prediction that stems from these results and from the pull-down experiments described above is that CsfB binds directly to σ E . To test this hypothesis, we purified His 6 -CsfB, σ E and σ K (both lacking their pro-sequences) overproduced in E. coli (Fig. 4B, first three lanes and S2A Fig) (Note that in addition to σ E , which has a molecular weigth of 29 kDa, a stable proteolytic fragment of about 20 kDa accumulated in the preparations; Fig. 4B). We then asked whether His 6 -CsfB immobilized onto a Ni 2+ -NTA column could capture untagged, purified σ E or σ K . While His 6 -CsfB bound efficiently to the Ni 2+ -NTA column, neither σ E nor σ K did (Fig. 4B). However, in the presence of His 6 -CsfB, σ E (but not σ K ), was efficiently retained by the column (Fig. 4B), a result fully consistent with the pull-down experiments described above (note that both σ E and the 20 kDa fragment were retained; Fig. 4B, left lanes).
To confirm these results with a different technique, we also carried out a GAL4-based yeast two-hybrid assay, an approach that had been used before to dissect the interaction between σ G and CsfB [20]. We constructed translational fusions of σ F (as a negative control), σ G (as a positive control), σ E , σ K , σ A or CsfB to the C-terminus of the GAL4 DNA binding (BD) or activation domains (AD). The various fusion proteins were expressed in different combinations in yeast cells and checked for their ability to interact in vivo, as assessed by the expression of a lacZ gene preceded by a GAL4-responsive element. As expected, we detected an interaction of CsfB with σ G , but not with σ F ( [20]; Fig. 4C). In addition, CsfB interacted with σ E (Fig. 4C), suggesting that the two proteins could indeed establish specific contacts. No interaction was detected between CsfB and σ K or σ A (Fig. 4C). Sigma-CsfB interactions. A: pull-down assays using GBP. Whole cell extracts were prepared for cultures of different strains 4 hours after the onset of sporulation in resuspension medium. The extracts were cleared and incubated with GBP bound to sepharose beads. Samples of the whole cell extracts, as well as the proteins bound to the GBP beads were visualized, following elution, by immunoblotting with anti-σ G , anti-σ E , anti-σ K and anti-GFP antibodies. Strains in A: a wild type strain, a strain producing GFP from the xylose-inducible P xylA promoter; strains producing CsfB-GFP from its native promoter region (P csfB ), from the σ F -type promoter (P sigF ) or the σ K -type promoter (P sigK ), as indicated. The asterisk indicates a likely degradation product of σ G . B: Ni 2+ -NTA affinity chromatography assay for CsfB-σ interactions. His 6 -CsfB, and untagged σ E and σ K , were purified from E. coli cells and analysed by SDS-PAGE (first three lanes). The three proteins were then individually applied to a column and eluted with an imidazole buffer. The eluted proteins were detected following SDS-PAG by Coomassie-staining. His 6 -CsfB bound to the Ni 2+ -NTA column whereas σ E or σ K did not. σ E but not σ K , bound to the column in the presence of His 6 -CsfB. The asterisk indicates a likely degradation product of σ E . Molecular weight markers (M, in kDa) are shown on the left side of panels B. The asterisks in A and B indicate likely degradation products of σ G or σ E . C: colony lift assay (left) and assays in liquid medium (right) for the detection of βgalactosidase activity in yeast strains expressing fusions of CsfB to the GAL4 activation domain (AD) and fusions of σ F , σ E , σ G , σ K , and σ A to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were expressed from empty vectors were used as negative controls ("-"). Thus, both the yeast two-hybrid and the affinity chromatography assays indicate that CsfB and σ E directly interact.

CsfB inhibits transcription in vitro from σ G -and σ E -dependent promoters
While CsfB binds to both σ G [20] and σ E ( [34]; see above), no study has shown direct inhibition of transcriptional activity by the anti-sigma factor. To test the ability of purified CsfB to inhibit σ G -or σ E -directed transcription, core RNA polymerase (E) was purified from B. subtilis, and reconstituted with σ F , σ E , σ G , σ K , or σ A overproduced and purified from E. coli cells (S2A Fig). As templates for in vitro transcription reactions, we used PCR-generated DNA fragments corresponding to promoters of genes known to be under the control of the sigma factors tested, and whose transcriptional start site has been mapped. As such, the gcaD gene was used as the template for σ A -directed transcription [35,36], spoIIQ as a template for Eσ F [27,37], spoIID, spoIIM, spoIIIA p1 and spoIIIA p2 as templates for Eσ E [38,39,40], sspB for Eσ G [30], and gerE for Eσ K [33] (Fig. 5A and S2B Fig). All forms of RNA polymerase tested directed the production of run-off transcripts of expected sizes (Fig. 5B-D and S2C Fig). No specific transcription products were seen when templates were mixed with core RNA polymerase in the absence of a σ subunit (S2C Fig). CsfB did not inhibit transcription by Eσ A , Eσ F (Fig. 5B) or Eσ K (Fig. 5C), but inhibited the σ E -directed utilization of the spoIIM (Fig. 5B), spoIID ( Fig. 5C and D), spoIIIA p1 and spoIIIA p2 promoters (S2C Fig). CsfB also inhibited the σ G -directed transcription of sspB ( Fig. 5B and D). Inhibition of Eσ A , Eσ F or Eσ K by CsfB was not observed even at molar ratios higher than those that inhibited Eσ E (Fig. 5C and D). Interestingly, inhibition of Eσ E , and to some extent of Eσ G , required molar ratios higher than 1 (Fig. 5B and D; S2C Fig). One possibility is that active CsfB is a dimer (or a higher order multimeric form), in agreement with the results of a previous study [21].
In total, the run-off transcription assays show that CsfB inhibits transcription by the RNA polymerases that contain the σ subunit to which it can bind in vitro, i.e., σ E or σ G .

Mapping the interaction between CsfB and σ E
To map the region(s) involved in the CsfB-σ E interaction, we performed GST-pull down and yeast two-hybrid assays, two techniques that were used before to map the interaction between CsfB and region 2.1 of σ G [20]. For that purpose, full-length mature σ E or different fragments of the sigma factor were fused to either GST or to GAL4 (Fig. 6A). For the GST pull-down assays, we used a CsfB-StrepII-tagged protein [20] and a GST-σ E fusion protein, lacking its pro region (sigma fragment a, Fig. 6A) or GST fusions to different fragments of σ E (fragments b to h; Fig. 6A), overproduced and partially purified from E. coli. We incubated CsfB-StrepII-tag with GST or the GST-σ E fusion proteins immobilized on glutathione beads. After washing, the presence of CsfB in the elution samples was assessed by immunoblotting with an anti-StrepII tag antibody. The CsfB protein was retained by GST-σ E (fragment a) but not by GST alone (Fig. 6B, left panel). GST (Fig. 6B). With the exception of fragment e (2.2/2.3, σ E 79-117 ), the same fragments also showed an interaction with CsfB in yeast two-hybrid assays (Fig. 6C). The lack of interaction of fragment e in the yeast two-hybrid assay may be due to the topology and/or stability of the GAL4 fusion.
Region 2.1 was previously implicated in the interaction of CsfB with σ G [20]. However, this region does not appear to be sufficient to mediate the interaction of CsfB with σ E . In contrast, because σ E 79-117 (regions 2.2/2.3) interacted with CsfB, but neither σ E 30-98 (regions 2.1/2.2) nor σ E 98-141 (regions 2.3/2.4) did, it is likely that residues at the end of region 2.2 and/or at the beginning of region 2.3 of σ E are necessary for the interaction. In any event, our results show that CsfB and σ E interact directly, and that the region required for the interaction differs from that found in σ G [20,22].  A single residue in region 2.3 allows CsfB to discriminate between σ E and σ K The N45E substitution in σ G results in loss of CsfB binding, whereas the E39N substitution in σ F is sufficient for conferring CsfB binding ability [20]. However, the residue homologous to N45 in σ E corresponds to a glutamate, E64 (Fig. 7A). Together with the mapping experiments described in the preceding section, this observation suggests that the interaction of CsfB with σ E differs from that with σ G . To more precisely delineate the determinants for CsfB binding to σ E , we sought to identify residues that are necessary for the interaction. An inspection of the amino acid sequence of σ E in regions 2.2 and 2.3 revealed an asparagine residue located at position 100 (i.e., at the beginning of region 2.3) and conserved among all of Bacillus σ E orthologues. Strikingly, the position homologous to N100 in σ K is occupied by an aspartate (E93) in B. subtilis and invariably (with the exception of the σ E proteins) by an acidic residue in all other Bacillus sigma factors (Fig. 7A). N100 in σ E is located in the vicinity of several residues involved in promoter melting (Fig. 7A). By contrast, E64 in σ E and N45 in σ G are located at the beginning of a helix within which several contacts are established with the β´subunit of RNA polymerase (S6 Fig). Thus, depending on which sigma factor is present in the complex, CsfB appears to bind to two distinct functional surfaces of the RNA polymerase holoenzyme.
We wanted to test whether the CsfB anti-sigma factor discriminated between σ E and σ K by differentiating an asparagine from an acidic residue at the beginning of region 2.3. We constructed GST and GAL4 fusions to forms of σ E bearing the single amino acid substitution N100E and used them in pull-down and yeast two-hybrid experiments. We found that GST-σ E N100E did not pull-down CsfB-strepII partially purified from E. coli cells (Fig. 7B) and showed a muchreduced interaction (when compared to the wild type sigma factor) with the anti-sigma factor in a yeast two-hybrid assay (Fig. 7C). By contrast, a GAL4 fusion to σ K bearing an E93N substitution interacted strongly with the anti-sigma factor in the yeast two-hybrid assay (Fig. 7C). To determine whether the N100E and E93N substitutions in σ E or σ K affected their activity in vivo, the corresponding mutations were first transferred to the sigE and spoIVCB genes (spoIVCB codes for the N-terminal half of σ K ; see the Material and Methods section for details). We then used P spoIVCA -(σ E -dependent promoter) and P cotG -lacZ (σ K -dependent promoter) transcriptional fusions to monitor the activity of σ E and σ K respectively, during sporulation. Consistent with loss of CsfB regulation, the N100E substitution increased expression of spoIVCA-lacZ, whereas the E93N replacement reduced expression of cotG-lacZ (Fig. 7D). Expression of P spoIVCA -lacZ or P cotG -lacZ in a ΔcsfB mutant did not differ much from the wild type (Fig. 7D), presumably because premature activation of pro-σ K somehow compensates for increased activity of σ E (see also above). In any event, the N100E substitution is sufficient to render σ E refractory to CsfB, whereas the E93N substitution suffices to make σ K susceptible to CsfB.

The N100E substitution in σ E affects the assembly of the spore surface layers
Assembly of a protective protein shell called the coat that forms the spore surface, involves the timely production of over 80 components under the control of σ E or σ K [41,42]. Since the N100E substitution increases the activity of σ E and reduces that of σ K we wanted to examine the coat protein composition in spores of the strain producing σ E N100E . Proteins were extracted from purified spores by treatment with a buffer containing SDS and the reducing agent DTT, or by NaOH [43]. The first treatment releases about 80 proteins from wild type spores (S7A Fig, top) whereas NaoH extraction produces a much smaller collection of extractable proteins (S7A Fig, bottom). We found that several proteins were more extractbale from N100E spores than from wild type spores, among which the species labelled a-e (SDS/DTT extraction) and f-k (NaOH extraction) in S7A Fig (bottom panel). For example, the outer coat proteins CotA (in band a), CotB (in b), and Tgl (in d), as well as the inner coat proteins CotN (in bands h and i), YaaH (in c) and YybI (in band d) were more extractable from σ E N100E spores (S7A Fig, see also S1 Text). Synthesis of YaaH, CotN and YybI is under σ E control, whereas production of Fig 7. A residue in σ E involved in the interaction with CsfB. A: alignment of regions 2.1, 2.2 and the beginning of region 2.3 from the σ A , σ F , σ G , σ E , and σ K proteins of the indicated species (the sequence of σ A from T. thermophilus is identical to that of T. aquaticus for the segments represented). The residue in B. subtilis σ G important for the interaction with CsfB is shown in a blue box. Note that the homologous position in σ F and in the other sigma proteins is invariantly occupied by a glutamic acid (yellow box). The residue herein identified in region 2.3 of B. subtilis σ E (N100) that is important for binding by CsfB is shown in a blue box. This residue is conserved among Bacillus orthologues of σ E whereas in other sigma factors the homologous position is occupied by and acidic residue (yellow box). Residues in region 2.2. of E. coli σ A implicated in core binding are highlighted in grey; residues, at the end of region 2.3, involved in promoter meting are also indicated (reviewed by [55]). The residues boxed in green were shown to affect sporulation in B. subtilis [20] and promoter melting in E. coli [68,69]. The accession numbers of the aligned sequences are given in the Material and Methods section. B: GST pull-down assays to investigate the role of E100 of σ E in the interaction with CsfB. GST, GST-σ E and GST-σ E N100E fusions were bound to glutathione beads and incubated with purified CsfB-Strep II (100 nM). Protein complexes were captured on glutathione sepharose beads, and visualized, following elution, by immunoblotting with anti-GST, or anti-Strep tag antibodies. C: colony lift assays (left) and assays in liquid medium (right) for the detection of β-galactosidase activity in yeast strains expressing fusions of CsfB to the GAL4 activation domain (AD) and fusions of σ E wt , or σ E N100E , σ K wt , or σ K E93N , to the GAL4 binding domain (BD), as indicated. Assays in which the BD and AD were produced from empty vectors were used as negative controls ("-"). D: effect of the sigEN100E and sigKE73N alleles on σ E -and σ K -dependent gene expression. The figure shows the expression of P spoIVCB -lacZ (σ E -dependent) and P cotG -lacZ (σ Kdependent) transcriptional fusions during sporulation. Samples were withdrawn from cultures of the represented strains, at the indicated times, in hours after the onset of sporulation in re-suspension (denoted as T0), and assayed for β-galactosidase activity (shown in Miller units). CotA, CotB, and Tgl is mainly controlled by σ K [9,29,41,44,45,46]. Thus, the N100E substitution has a strong and global impact on the assembly of the spore surface layers, affecting the assembly of proteins from both the inner and outer coat layers, which are produced at different periods (controlled by σ E or by σ K ) during spore coat assembly.

Discussion
A key finding of our study is that expression of csfB switches from the forespore to the mother cell midway into the process of sporulation. The regulatory region of csfB includes two promoters: a σ F -type promoter (P sigF ) utilized by σ G in pre-divisonal cells and by σ F in the forespore [14,17,20], and a σ K -type promoter (P sigK ), located further upstream, active in the mother cell following engulfment completion. Activation of csfB transcription in the mother cell coincides with a decline in forespore-specific expression (Fig. 2). It has been hypothesized that a reduction in the levels or activity of CsfB in the forespore could be a factor promoting σ G activity following engulfment completion [14,17,20]. SpoIIAB also disappears from the forespore following engulfment completion [47]. It remains unclear how CsfB activity is turned off in the forespore during the late stages of sporulation, since σ G can utilize P sigF in vitro and in vivo ( [20]; this work). However, it has been suggested that Zn 2+ -depletion in the forespore, following engulfment completion, could render CsfB unstable [21]. If so, then the decrease in the CsfB-GFP signal in the engulfed forespore (Fig. 1B) could represent protein degradation.

Enforcing the transition from early to late stages of development
Genome-wide transcriptional profiling analysis showed that inactivation of P sigK , which abolishes CsfB accumulation in the mother cell, caused increased transcription of σ E -controlled genes that either rely solely on σ E for expression or are dependent on the type I coherent FFL formed by σ E and the ancillary transcriptional activator SpoIIID [9]. Increased σ E activity was also observed using a σ Eresponsive lacZ reporter in a strain expressing the CsfB-resistant form of σ E (σ E N100E ). By contrast, σ E -dependent genes repressed by GerR or SpoIIID (i.e., the output of the type I incoherent FFLs generating pulses X 2 and X 3 in Fig. 1B) did not show increased expression in the P sigF -csfB strain, most likely because their transcription has already been switched off at the time of analysis due to the action of the two repressors (Fig. 3A). In general, these observations are consistent with an increase in the activity of σ E in the absence of CsfB and with the properties of the FFLs formed by σ E [3]. They also support a model in which the appearance of CsfB in the mother cell promotes the transition from early to late cell-type specific gene expression (i.e., the σ E to σ K switch) (Fig. 8A).
While a previously described negative feedback loop driven by σ K reduces the levels of σ E [11,12,48], the feedback loop revealed in this work, and similarly initiated by σ K , uses CsfB to limit the activity of σ E in the mother cell. Thus two partially redundant feedback loops evidently function to promote proper switching from σ E -to σ K -dependent transcription following engulfment completion, and thus the developmental transition from early to late stages of morphogenesis (Fig. 8A). σ G and the activity of σ K Interference with csfB expression in the mother cell, as when P sigK is inactivated, also leads to increased expression of several genes under σ K control (Fig. 3). Because CsfB does not bind to σ K and does not inhibit σ K -directed transcription in vitro, it is unlikely that the increased activity of σ K is due to the loss of a direct interaction with CsfB. Instead, it is more likely to be a consequence of the ectopic activation of σ G in the mother cell. Previous work has shown that two negative regulators of σ G , the anti-sigma factor SpoIIAB and the LonA protease, effectively counteract σ G in the mother cell, when its synthesis is artificially induced by driving expression of sigG from a σ E -dependent promoter [15]. Under the same genetic conditions, we showed that CsfB contributes, along with LonA and SpoIIAB, to the inhibition of σ G activity in the mother cell (S5 Fig). In the P sigF -csfB mutant, our transcriptional profiling data indicate that most of the genes in the σ G regulon show increased expression. This increased expression is due in part to increased activity of σ G in the mother cell of P sigF sporangia. Not only transcription from the σ G -dependent P sspE promoter fused to cfp is detected in the mother cell prior to engulfment completion (S4 Fig), but FISH experiments also show accumulation of the sspE transcript in the mother cell (Fig. 3). Therefore, CsfB is one of several redundant mechanisms that act to silence σ G in the mother cell.
In wild type cells, it is the activity of σ G in the forespore following engulfment completion that triggers σ K activation in the mother cell, via the signalling protein SpoIVB. Mutations that bypass this signalling pathway result in premature activity of σ K and defects in spore morphogenesis [25]. We have shown that the production of σ G in the mother cell uncouples pro-σ K processing from engulfment completion and leads to premature activation of σ K because SpoIVB can activate processing of pro-σ K directly in the cell where it is produced ( S5 Fig). Accordingly, processing of pro-σ K in the P sigF mutant is detected earlier than in the wild type (Fig. 3C). By contrast, in the σ E N100E strain, expression of a σ K -dependent lacZ fusion is reduced and delayed, presumably because pro-σ K processing remains strictly dependent on the post-engulfment, σ G -dependent SpoIVB signalling from the forespore. Therefore, the silencing of σ G in the mother cell by CsfB, along with the contributions of SpoIIAB and LonA, prevents premature activity of σ K .

Discrimination by CsfB of the σ F / σ G and σ E / σ K pairs
We have previously shown that N45, an asparagine residue in region 2.2 of σ G , is a critical determinant for CsfB binding [20] (Fig. 1C). Not only is this residue conserved among Bacillus orthologues of σ G but a glutamic acid residue, E39, is found at the homologous position of the CsfB-resistant σ F protein in B. subtilis, and an acidic residue is invariably found at the equivalent position in σ F orthologues of other Bacillus species. Importantly, the N45E substitution renders σ G refractory to CsfB binding and conversely, the E39N variant of σ F is susceptible to the anti-sigma factor. Residue N45 of σ G is most likely involved in a direct contact with the βś ubunit of RNA polymerase, suggesting that CsfB, like other anti-sigma factors interferes with the σ/β´interaction [20]. Our in vitro transcription assays support this model, as CsfB is sufficient to inhibit σ G -and σ F -directed transcription at bona fide promoters in vitro (Fig. 5).
Our results now implicate a region encompassing regions 2.2 and 2.3 of σ E in CsfB binding (Fig. 6), and an asparagine residue, N100, at the beginning of region 2.3, was found to be a key determinant (Fig. 7A-C). In a striking parallel with the σ F /σ G pair, a homologous asparagine is found in all known orthologues of σ E from related organisms (Fig. 7A). Despite the high degree of similarity between the σ E and σ K proteins across sporeformers [4,5,6,7], the homologous residue in the σ K protein of B. subtilis is a glutamic acid, E93, and an acidic residue is invariably found among Bacillus orthologues of σ K (Fig. 7A). Also reminiscent of the σ F /σ G pair, the N100E substitution makes σ E refractory to CsfB, whereas the E73N variant of σ K becomes susceptible to the anti-sigma factor (Fig. 7B to D).
Thus, binding of CsfB to any of the four sigma factors of sporulation is favored by the presence of a conserved asparagine in region 2.1 (for σ F /σ G ) or 2.3 (σ E /σ K ) and hindered by a glutamic acid at either position (Fig. 8B). Resistance to CsfB binding requires both positions to be occupied by acidic residues. This discrimination is essential for the control of gene expression during sporulation: it allows σ G activity to be inhibited in the forespore prior to engulfment completion, while allowing σ F -dependent transcription; it also enables CsfB to antagonize σ E and σ G in the mother cell thus enforcing the cell type-specificity of σ G and facilitating the timely switching from σ E to the CsfB-immune σ K . Making σ F or σ K susceptible to CsfB or making σ E or σ G resistant to the anti-sigma factor interferes with proper temporal control and compartmentalization of the forespore and mother cell lines of gene expression.
The strict conservation of the discriminating residues in regions 2.1 or 2.3 of the sigma factors among Bacillus species (Fig. 7A) underscores the importance of CsfB for the activity of the cell type-specific sigma factors in sporeforming organisms closely related to B. subtilis.
Binding of CsfB to σ F /σ G and to σ E / σ K The crystal structure of the σ 70 -containing RNA polymerase holoenzyme from Thermus aquaticus shows that residue E189, the homologue of N45 in σ G , is involved in a direct contact with K159 in the β´subunit [49,50,51,52]. We have argued that an asparagine residue could also contribute to the interaction with this site of β´, consistent with the view that one mechanism by which anti-sigma factors function is by occluding sigma-core binding interfaces [53,54]. Occluding a β´-binding surface is consistent with a role for CsfB in preventing the ectopic activity of σ G in pre-divisional cells [20] or the premature activation of σ G in the forespore [14]. In contrast, N100 in σ E is located in the beginning of region 2.3, just upstream of a motif containing several conserved residues involved in promoter melting [55] (Fig. 7A and S6 Fig). This suggests that CsfB may interfere with σ E function at a step during transcription initiation subsequent to closed complex formation. In contrast to σ G , whose ectopic or premature activation Model for the functions of CsfB. A: following engulfment completion, two negative feedback loops are established that act to limit the level (brown line) and the activity of σ E (red line). The loop that acts at the level of σ E activity involves the σ K -dependent production of CsfB (this work), and restricts mainly the expression of the σ E -and SpoIIID-dependent gene class. Both feedback loops promote proper switching from the early (pre-engulfment, σ E -dependent) to late (post-engulfment completion, σ K -dependent) stages in development. Together with LonA and SpoIIAB, CsfB also antagonizes σ G in the mother cell (grey line). Minimizing the chances of σ G becoming active in the mother cell prevents the premature, forespore-independent activation of σ K . Thus, the action of CsfB superimposes both onto the transcriptional and cell-cell signaling networks. B: the panel represents the residues that allow binding of CsfB to σ G (N45) and σ E (N100), and that are sufficient to render σ F (E39) and σ K (E93) resistant to the anti-sigma factor. N45 in σ G and E39 in σ F are located in conserved region 2.1 (purple sector) whereas N100 in σ E and E93 in σ E are located in region 2.3 (yellow). has to be prevented, σ E is already engaged in transcription when CsfB appears in the mother cell. Therefore, the most effective mechanism for antagonizing σ E activity may not be targeting a core-binding surface but rather a functional region that prevents the activity of the σ E -containing RNA polymerase holoenzyme. We note, however, that N100 is also close to several residues in region 2.2 that have been implicated in core binding in E. coli σ 70 , and are conserved in B. subtilis σ A (highlighted in grey in Fig. 7A) (reviewed by [55]). Thus, binding of CsfB in the vicinity of N100 could also possibly occlude core-binding sites in region 2.2.

Concluding remarks
The function of CsfB affects both the transcriptional and the cell-cell signalling networks that control spore differentiation (Fig. 8A). In the forespore, CsfB contributes to the inhibition of σ G during early stages of development [14]. The σ K -dependent expression of csfB in the mother cell limits the activity of σ E and prevents the ectopic activation of σ G These activities of CsfB are each one part of redundant mechanisms that work to the same end. σ K activity blocks expression of sigE by an unknown mechanism [11,12], and ectopic activity of σ G is limited by SpoIIAB and LonA in the mother cell. Our discovery that CsfB binds and inhibits the activity of σ E also leads us to propose that in the forespore, CsfB binding to σ E explains how CsfB functions as one of the several partially redundant mechanisms described by Piggot and co-workers [34] that prevent ectopic activity of σ E in the forespore (see also Fig. 1B). That several of the major roles for CsfB (i.e., preventing ectopic expression of σ G and σ E , and facilitating the transition between σ E and σ K ) involve partially redundant mechanisms speaks to the importance of controlling these processes, and provides an explanation for why CsfB is highly conserved among sporeformers [6,21]. Moreover, these redundancies may also explain why the phenotype of a csfB mutant is relatively mild. The original work on CsfB found no decernable phenotype [23], whereas subsequent work from Stragier's group [22] described a small but significant germination defect, but only when a ΔcsfB mutation was combined with an allele causing premature transcription of the gene for σ G [22]. The P sigF and P sigK strains formed spores with the same efficiency (77% and 88%, respectively) as the wild type strain (83%). However, we did observe a difference in the protein composition of the spore surface layers between σ E N100E and wild type spores (S7A Fig). It is unknown whether this phenotype provides some of the selective pressure for maintaining CsfB during the evolution of sporeformers, but we note the important role of the spore surface layers in mediating many of the environmental interactions of spores, including with cells of host organisms [42]. On the other hand, the evolution of redundant mechanisms to control key steps in development may have promoted the stabilization or canalization of the cell type-specific patterns of gene expression that led to the establishment of the endospore differentiation pathway [56].
The role of CsfB in favouring the switch from early to late stages in spore development is likely to be conserved among Bacillus species and other sporeformers. Importantly, at least in Bacillus species, the actions of the CsfB anti-sigma rely strongly on its ability to discriminate between the highly similar σ F /σ G and σ E /σ K pairs (Fig. 8B). Interestingly, CsfB is not found in some Clostridia, a more distantly related class of sporeforming organisms, including the human intestinal pathogen C. difficile. The gene regulatory network for sporulation has been recently characterized in detail for C. difficile. It is interesting to note that in this organism, σ G is active prior to engulfment completion and that σ E remains active until the late stages of sporulation [57,58,59]. The looser temporal control of σ factor function in C. difficile and presumably also in other Clostridia may stem, at least in part, from the absence of a CsfB orthologue. Moreover, it may be interesting to investigate whether this looser regulation results in greater heterogeneity of morphology among spores of certain Clostridial species than among spores of Bacillus species.

Strains and general techniques
Except for strain MBS3656 ( [60]; see below), all other B. subtilis strains used in this work are congenic derivatives of the Spo + strains MB24 (trpC2 metC3) or PY79 (prototrophic). Their construction is detailed in S1 Text, and they are listed, with their genotypes, in S1 Table. All plasmids used in this work are described in S1 Text. Primers used for plasmid construction, mutagenesis or sequencing are listed, with their sequences, in S2 Table. LB medium was used for routine growth or maintenance of E. coli and B. subtilis, and sporulation was induced by growth and exhaustion in Difco sporulation medium (DSM) or by the re-suspension method [43].
Purification of RNA polymerase, CsfB and the sigma factors σ A , σ F , σ E , σ G and σ K The strains for sigma factor and CsfB expression were cultured in LB at 37°C with Ampicillin (100μg/ml). Arabinose was added to final concentration of 0.5% at an OD 600 of 0.6. The culture was allowed to incubate for another 1-2 hours before cells were harvested and stored at -80°C. Sigma factors and CsfB were purified using Qiagen Ni-NTA. The Ni-NTA-affinity purified protein was analyzed on a 10% SDS-PAGE. Fractions containing the sigma factor or CsfB were pooled and dialyzed against dialysis buffer (50mM Tris, 100mM NaCl, 3mM β-mercapatoenthanol). The dialyzed protein was chromatographed through a GE HiLoad 16/60 Superdex 75 column, fractions (1 ml) collected, and those containing the desired protein pooled and dialyzed against 1X in vitro transcription reaction buffer (50mM Tris, 100mM KCl, 10% Glycerol, 10mM DTT). Protein concentration was measured using a Bradford assay (Bio-Rad, Hercules, CA). Protein aliquots were stored at -20°C.
RNA polymerase (RNAP) was purified from B. subtilis strain MH5636 [60] cultured in LB with chloramphenicol (5 μg/ml) to an OD 600 of 1.0. Cells was harvested by centrifugation and lysed by treatment with lysozyme (5mg/ml) for 30 minutes at 4°C followed by passage twice through a French pressure cell at a pressure of 20,000 psi. Core RNA polymerase was purified essentially as described by Burgess and colleagues [55]. Briefly the cell lysate supernatant was first purified using Qiagen Ni-NTA affinity column. Fractions containing RNAP were pooled and subjected to chromatography on a HiLoad 16/60 Superdex 200 gel filtration column. Fractions containing RNA polymerase were then purified by ion-exchange exchange chromatography on a GE Mono Q 5/50 GL column. The purity of RNAP was assayed on a 4-20% SDS-PAGE. The concentration of purified RNAP was determined using a Bradfors assay (Bio-Rad, Hercules, CA). RNAP aliquots were at -20°C.

In vitro transcription assays
Core RNAP (13 nM) was incubated in transcription reaction buffer (40 mM Tris/HCl (pH 8), 50 mM KCl, 10 mM MgCl2, 10mM DTT, 50 mg/ml acetylated BSA, 0.5 ml RNase-inhibitor [61] on ice for 30 minutes with 130 nM sigma factor and 1.0 μg of a DNA template purified through a CsCl/ethidium bromide density gradient and cleaved with a restriction enzyme (BamHI or HindIII). After 10 min at 37°C, ATP, CTP, and GTP were added to a final concentration of 1.0 mM and 5 μCi UTP was added to 50 μl transcription reactions. The mixture was incubated for 10 minutes at 37 7°C after which 0.2 mM unlabeled UTP was added, followed by a further incubation for 10 minutes at 37°C. Finally, the reaction mixture was extracted with phenol-chloroform and the nucleic acids precipitated and electrophoretically resolved through 10% (w/v) polyacrylamide gels containing 7M urea. In the reactions containing CsfB, the anti-sigma factor was added at an amount equal to sigma (130 nM) or 2, 3, 4 or 8-fold excess (indicated as 1x to 8x in Fig. 6C and D), and incubated with sigma for 20 min at room temperature. RNAP was added and the reactions conducted as described above, and incubated with sigma for 20 min at room temperature. RNAP was added and the reactions conducted as described above.

Transcriptional profiling of sporulating cells
These analyses were carried out as previously described in Cozy et al. [62] and Winkelman et al. [63]. Briely, the arrays obtained form Agilent include 15,744 probes covering the annotated protein-coding genes of B. subtilis [64] and the small non-coding RNAs reported in Rasmussen et al. [65] and Irnov et al [66]. The arrays were designed using the Agilent eArray application. Strains AH6825 (P csfB -csfB) and AH6827 (P sigF -csfB) were sporulated by resuspension in Sterlini-Mandelstam medium and samples collected at hour 3 of sporulation. Cells pellets were recovered by centrifugation after mixing with an equal volume of cold methanol. Total RNA was recovered using a hot acid-phenol protocol followed by clean-up using the Qiagen RNeasy kit. cDNA was synthesized from the purified total RNA and labelled using the Agilent Fairplay III kit. After hybridization and washes using standard protocols, the arrays were scanned in an Agilent Technologies DNA microarray scanner with Surescan high-resolution technology. Processed signal from the Agilent software was subjected to standard lowess normalization using Bioconductor run in R and the geometric mean of the probes was used to give the final value for each gene and nc-RNA.

Fluorescence in situ hybridization (FISH)
For RNA-FISH, we used the following protocol: cells growing in Re-suspension medium were fixed in Histochoice solution (Ameresco) for 15 min at room temperature and 30 min on ice. The samples were centrifuged three times at 3.000xg for 2 min and washed in 1xDEPC-treated PBS. The cell pellets were resuspended in 100 μl GTE buffer (50 mM glucose, 20 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8). Sixteen microlitres of a 10 mg/ml lysozyme solution (GTE, 4 mM vanadyl ribonucleoside complex (VRC), 10 mg/ml lysozyme) were added to 48 μl of cell suspension. The mixture was immediately placed onto poly-L-lysine-coated multi-well slides, and incubated for 10 min at room temperature. The excess liquid was aspirated and the slides were left 1 min to dry before putting them in -20°C methanol for 10 min. Next, the slides were dipped in -20°C acetone for 30 s. Once the slides were dry, they were incubated at 37°C for 30-60 min in a 40% formamide solution (40% formamide, 2x DEPC-treated saline-sodium citrate buffer (SSC)). LNA probe was added to the hybridization solution I (80% formamide, 1 mg/ml E. coli tRNA, 2x DEPC-treated SSC, 70 μg/ml calf-thymus DNA) at a final concentration of 250 nM, and incubated at 80°C for 5 min before mixing with the hybridization solution II (20% dextran sulphate, 4mM VRC, 40U RNase inhibitor, 0.2% RNase-free BSA, 2x DEPC-treated SSC) in a 1:1 ratio. The hybridization solution (25 μl) was added to each well of the slide and hybridized for 2 h. The slides were then washed twice in 50% formamide and 2x DEPC-treated SSC solution for 30 min and briefly rinsed five times in DEPC-treated PBS. DAPI was added to a final concentration of 10 μg/ml in SlowFade solution (Invitrogen) were added to each well and the slide was covered and sealed using clear nail polish. The slides were either visualized immediately or stored in the dark at -20°C. The sequence of the probes, which were labeled with Cy3, is given S2 Table. Fluorescence microscopy Samples (0.6 ml) of cultures were collected, resuspended in 0.2 ml of phosphate-buffered saline (PBS) and the membrane dye FM4-64 (Molecular Probes) added to a final concentration of 10 μg ml −1 . Cells were observed on slides padded with 1.7% agarose. Images were acquired on Leica fluorescence microscopes DMR2A and DM6000B equipped with a Cool Snap HQ camera (Roper Scientific, Arizona, USA) and an iXonEM+ 885 camera (Andor Technology, Connecticut, USA), respectively, or on a Nikon E1000 microscope equipped with an Orca-ER camera (Hamamatsu Corporation, New Jersey, USA), using 63x or 100x lens objective plus an additional 1.6X optavar, phase-contrast optics and standard filters for visualization of GFP and FM4-64. Images were acquired using Metamorph (Molecular Devices, Berks, UK) and processed for publication using Photoshop (Adobe).

Accession numbers
The Genbank accession numbers for the sequences represented in Fig. 8A and S3C Fig, are  Supporting Information S1 Fig. csfB expression during sporulation. A: immunoblot analysis of CsfB-GFP accumulation during sporulation. Samples from sporulating cultures were collected at the time of ressuspension in resuspension medium and at hourly intervals thereafter, as indicated by the numbers above the lanes. Whole cell extracts were prepared and the proteins (30 μg samples) were resolved on 15% SDS-PAGE gels and subject to immunoblot analysis with an anti-GFP antibody. Arrows mark the position of CsfB-GFP. B: expression of transcriptional csfB-lacZ fusions inserted at the amyE locus during sporulation. The following strains were included in the analysis: a wild type (bearing no lacZ fusion, to estimate background levels), a fusion of the σ Ftype csfB promoter to lacZ in the wild type background and in a sigG deletion mutant, a fusion of the σ K -type csfB promoter to lacZ in the wild type background and in a sigK deletion mutant. Cultures of the various strains were induced to sporulate by the resuspension method, and samples withdrawn at the indicated times, in hours, after the onset of sporulation (denoted as T0), and assayed for β-galactosidase activity (shown in Miller units). shown is a 15% SDS-PAGE gel separation of core RNA polymerase purified from B. subtilis cells (E), and CsfB, σ E , σ F , σ G , σ K , and σ A (arrows) overproduced and purified from E. coli. Asterisks indicate possible degradation products. The position of molecular weight markers (M, in kDa) is shown on the left side of the panel. B: schematic representation of PCR products bearing the control regions and part of the coding sequence of the spoIID and spoIIIA genes, used as templates for in vitro transcription reactions with RNA polymerase containing σ E and σ K , respectively. The size of the expected run-off products is indicated in nucleotides. C: in vitro transcription reactions with RNA polymerase containing σ E and the spoIID, spoIIIA P 1 or spoIIIA P 2 templates in the absence ("-") or in the presence of varying concentrations of CsfB (indicated as molar ratio relative to RNA polymerase). The arrows, color-coded as the templates in panel B, point to the position of the resulting run-off products. The position of molecular weight markers (in nucleotides) is shown on the left side of the panel. (TIF) S3 Fig. Mutations in the σ F -dependent csfB promoter leads to σ G activity in pre-divisional sporangia. A: shows the effect of csfB mutations on the activity of σ G , monitored by means of an sspE-lacZ transcriptional fusion. Cells harbouring the indicated mutations were grown in Difco sporulation medium (DSM) and samples collected at the indicated times, in hours, before or after the onset of stationary phase (or T0), and assayed for β-galactosidase activity (shown in Miller units). B: shows the effect of csfB mutations on the activity of σ G , monitored by means of an sspE-cfp transcriptional fusion. Cells were grown in DSM, samples collected 2 hours after T0, stained with the membrane dye FM4-64 and examined by fluorescence microscopy. Cells showing no signs of asymmetric septation and a strong CFP signal are indicated by yellow arrows. The numbers in the CFP panels indicate the percentage of cells with a similar pattern of CFP fluorescence. Scale bar, 1 μm. (TIF) S4 Fig. In a P sigF -csfB mutant, expression of sspE takes place in the mother cell, prior to engulfment completion. A: strains carrying a transcriptional fusion of the σ G -dependent P sspE promoter to cfp in either the wild type or P sigF -csfB backgrounds were indiced to sporulate by the re-suspension method. Samples were collected 3 hours after re-suspension, the cells stained with the membrane dye FM4-64 and examined by fluorescence microscopy. Yellow or white arrows point the fluorescence signal in the forespore (following engulfment completion) or in the mother cell (prior to engulfment completion, as judged from the absence of forespore labeling by the FM4-64 dye). B: quantification of the fluorescence signal obtained for the wild type or P sigF -csfB strains expressing P sspE -cfp. The signal was only measured in the mother cell, in cells that had not completed forespore engulfment. Fluorescence intensity is shown in arbitrary units. (TIF) S5 Fig. CsfB concurs with SpoIIAB and LonA to antagonize σ G in the mother cell. A: shows the effect of insertional mutations in lonA or csfB, or a point mutation in the gene for σ G (sigG E156K) on the activity of σ G (monitored by means of an sspE-lacZ fusion) when produced in the mother cell from the spoIID promoter. B: the panel illustrates the expression of a fusion of the σ K -dependent gerE promoter to lacZ, during sporulation, in strains expressing either sigG or sigG E156K from the mother cell-specific spoIID promoter. In A and B, cultures were grown in DSM, samples withdrawn at the indicated times in hours after the onset of sporulation (denoted as T0), and assayed for β-galactosidase activity (in Miller units). All strains, in A and B, carry an in-frame deletion of the sigG gene and a copy of sigG or sigG E156K under P spoIID or P sigG control, inserted at the amyE locus. C: illustrates the activation of the pro-σ K activation complex (red circle) by SpoIVB, produced in the mother cell, and the role of SpoIIAB, LonA and CsfB in reducing the potential for σ G activity in the mother cell. The composite negative feedback loop established by CsfB and σ G is similar to the one that limits the activity of the sigma factor in pre-divisional cells [20]. Transcriptional and protein-protein interactions are shown in black and red, respectively. (TIF) S6 Fig. Crystal structure of σ A -containing RNA polymerase. A: The figure shows the crystal structure of σ A -containing RNA polymerase holoenzyme from T. thermophilus (pdb: 1IW7), with the residues homologous to N45 in B. subtilis σ G (E202) and E100 in σ E (E240) highlighted in stick representation. The two α subunits are represented in magenta and purple, β is shown in grey, β´in green, and σ in yellow. B: expansion of the region encircled in A. Note that E202 forms a salt bridge with R159 in β´, whereas E240 is surface exposed. The image was rotated 180°along the y axis to generate the bottom panel. The images were rendered with Pymol (www.pymol.org). C: The figure shows an alignment of the primary structures of a segment of the β´subunit of RNA polymerase from B. subtilis, E. coli, T. aquaticus and T. thermophilus. E202 in T. thermophilus and T. aquaticus σ A (in region 2.1) and presumably N45 in B. subtilis σ G make a contact with the Arg residue marked by a dot in the β´subunit of RNA polymerase. Note that this residue is not conserved in the β´subunit from E. coli (see also [20]). The T. aquaticus holoenzyme is not show in panels A and B but is highly similar to the T. thermophilus enzyme, and the relevant residues in σ and β´are identical. The alignment was produced with ClustalW (www.ebi.ac.uk) and the accession numbers given at the end of the Material and Methods section. (TIF) S7 Fig. The N100E mutation in sigE affects the assembly of the spore surface layers. A: spores of the indicated strains were purified on density gradients and proteins extracted by treatment with an SDS/DTT-containing buffer (top) or NaOH (bottom). The red arrows indicate the position of proteins that are more extractable from spores produced by the strain producing the N100E form of σ E (see also S1 Text). B: spores of the indicated strains, identified by the color code used in panel A, were induced to germinate by exposure to L-Alanine (10 mM; close symbols). Spores in control samples (open symbols) were heat activated but not exposed to L-Alanine. The rate and extent of germination monitored by following the drop in the optical density of the suspension (OD) at 580 nm, over time, and expressed as the percentage of the initial OD 580 . (TIF) S1 Text. Supplemental materials and methods and supplemental results and discussion. (DOCX) S1 Table. Bacillus subtilis strains used in this work (PDF). (DOCX) S2 Table. Oligonucleotides used in this work (PDF). (DOCX) S3 Table. Transcriptional profiling of sporulating cells (PDF). (PDF)