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J Bacteriol. Oct 2005; 187(19): 6832–6840.
PMCID: PMC1251595

Control of the Expression and Compartmentalization of σG Activity during Sporulation of Bacillus subtilis by Regulators of σF and σE

Abstract

During formation of spores by Bacillus subtilis the RNA polymerase factor σG ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of σG ordinarily requires prior activity of σF in the prespore and σE in the mother cell. Here we report that in spoIIA mutants lacking both σF and the anti-sigma factor SpoIIAB and in which σE is not active, σG nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of σG activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for σG, from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to σG. When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, σG became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature σG activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of σG activity, and pro-σE (but not σE) can counteract this effect of SpoIIGA. We find that in strains lacking σF and SpoIIAB and engineered to produce active σE in the mother cell without the need for SpoIIGA, σG also becomes active in the mother cell.

Central to cell differentiation is the establishment of distinct programs of gene expression in the different cell types involved. These programs determine the subsequent path of differentiation. Among prokaryotes, formation of spores by Bacillus subtilis has become a paradigm for the analysis of cell differentiation. Soon after the start of spore formation, bacteria divide asymmetrically to give the smaller prespore (also called the forespore) and the larger mother cell. The prespore is then engulfed by the mother cell. The prespore develops into the mature spore, whereas the mother cell ultimately lyses. The process of spore formation is characterized by the cell-specific activation of four RNA polymerase σ factors. Immediately after the completion of the spore division septum, σF is activated in the prespore. Its activation leads rapidly to activation of σE in the mother cell. Upon completion of engulfment, σG becomes active in the prespore; its activation, in turn, leads to activation of σK in the mother cell (Fig. (Fig.1)1) (reviewed in reference 10). The activation of the successive σ factors is tightly coordinated within and between the two cell types, a process that has been termed crisscross regulation (19). We explore here the activation of σG in circumstances in which its normal tight coupling to the prior activation of σF and σE has broken down.

FIG. 1.
Schematic representation of stages of spore formation showing the normal location of activity of sporulation-specific sigma factors.

Both σF and σE are formed soon after the start of spore formation and before the sporulation division. When first formed they are inactive: σF because of interaction with the anti-sigma factor SpoIIAB and σE because it is formed as an inactive precursor, pro-σE. A complex regulatory system centered on SpoIIAB controls the activation of σF, which occurs in the prespore shortly after completion of the sporulation division. Activation of σE in the mother cell by processing of pro-σE depends on SpoIIGA, which is the putative processing enzyme, and on a σF-directed signal from the prespore. The appearance of σG activity depends on the activities of both σF and σE and on morphological signals (10).

The spoIIIG locus, which encodes σG, is first transcribed early in sporulation by read-through from the upstream spoIIG locus (Fig. (Fig.2).2). However, there is little, if any, translation of this transcript, probably because the spoIIIG ribosome-binding site is sequestered in a stem-loop structure; further, the transcript is not necessary for spore formation (20, 35). Following septation, the spoIIIG locus is transcribed productively from its own σF-directed promoter (6, 13), which is active exclusively in the prespore (reviewed in reference 25). Transcription from that promoter, which also depends on a σE-directed signal from the mother cell (13, 21), leads to the formation of σG (35). When first formed, σG is inactive; additional signals are required to activate it. Activation of σG requires expression of spoIIIJ in the prespore and of spoIIIA in the mother cell, and these are thought to act via a direct regulator of σG that has yet to be identified (30). Activation also requires completion of engulfment of the prespore by the mother cell (33). SpoIIAB can act as an anti-σ for σG as well as for σF (5, 15, 30) but is now thought not to be a regulator of σG activity in the prespore (32).

FIG. 2.
Schematic representation of the spoIIG-spoIIIG region of the chromosome. The promoter for spoIIG requires σA and activated Spo0A for expression. The promoter specific to spoIIIG requires σF or σG for expression. The different transcripts ...

Here we explore determinants of σG regulation. We find that in the absence of both σF and the anti-sigma factor SpoIIAB, σG becomes active in the mother cell instead of the prespore. Further, activation follows completion of septation rather than completion of engulfment. We describe a previously unrecognized control, in which SpoIIGA can prevent the appearance of σG activity, and pro-σE can counteract this effect of SpoIIGA. We also find that premature activation of σG can prevent septum formation.

MATERIALS AND METHODS

Media.

B. subtilis was grown in modified Schaeffer's sporulation medium (MSSM) or on Schaeffer's sporulation agar (23, 28). When required, the medium contained chloramphenicol at 5 μg/ml, erythromycin at 1.5 μg/ml, neomycin at 3.5 μg/ml, spectinomycin at 100 μg/ml, or tetracycline at 10 μg/ml. Escherichia coli was grown on LB (Luria-Bertani lysogeny broth) agar containing ampicillin at 100 μg/ml when required.

Strains.

B. subtilis 168 strain BR151 (trpC2 metB10 lys-3) was used as the parent strain. B. subtilis strains used are listed in Table Table1.1. The spoIIAΔ4 mutation was described previously (24); the deletion encompassed the entire spoIIA operon, but the ends of the deletion have not been defined. In the spoIIAΔ::neo and spoIIAΔ::spc mutations, the entire spoIIA operon, from 48 bp upstream of the first open reading frame (ORF) to 7 bp downstream of the last ORF, was replaced with the antibiotic resistance cassette. In the mutation designated spoIIAB-ACΔ::neo, the entirety of spoIIAC and all but the 5′ 163 bp of spoIIAB were replaced with a neo cassette. In the spoIIACΔ::neo mutation, 543 bp from the 3′ end of the spoIIAC ORF were replaced with neo. The spoIIGBΔ::spc mutation was derived from EU8701 of Kenny and Moran (16). The spoIIGΔ::cat mutation has 388 bp from the 3′ end of spoIIGA and 338 bp from the 5′ end of spoIIGB replaced with the cat cassette. The spoIIG(P)Δ::cat mutation has the region from 142 bp upstream of spoIIGA (including its promoter) to 388 bp into spoIIGB replaced with the cat cassette. The spoIIGA::cat mutation has the cat cassette inserted at the StuI site located 389 bp from the 3′ end of the ORF. The gene for the pro-less form of σE, sigE, was inserted at thrC under the control of the spoIIG promoter; in the encoded protein, N-terminal MH residues are joined to residue 28 (Y) (pro-σE numbering). Strain AH2487, containing a translational σG-green fluorescent protein (GFP) fusion, was kindly provided by Adriano Henriques. DNA from that strain was used to introduce the fusion into BR151 (spo+) to yield SL12673 and into a spoIIAΔ::neo derivative of BR151 to yield SL12674. The spoIIIG::neo mutation has the resistance cassette inserted in the Pst1 site within spoIIIG. DNA containing the amyE::spoIIIG construct (35) was kindly provided by Peter Setlow and DNA with the lonA disruption by Adriano Henriques. The PspoIIE-spoIIR construct was described by Zhang et al. (38). The Pspac(hy) vector of Quisel et al. (27) was used to place the entire spoIIGB or spoIIGA ORF, with its ribosome-binding site, at thrC under IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible control. E. coli DH5α (Gibco-BRL) was used to maintain plasmids. Details of strain construction are available on request.

TABLE 1.
B. subtilis strains used

Fluorescence microscopy.

Cultures were grown in MSSM at 37°C. A 200-μl volume of culture was mixed with 2 μl of FM4-64 (Molecular Probes) that had been previously diluted to 1 mg/ml in phosphate-buffered saline (Gibco-BRL). Samples were incubated at 37°C for 5 min, and 1 μl of unfixed sample was transferred to a slide and visualized essentially as described by Pogliano et al. (26). Images were captured using a Leica DM IRE2 microscope with a TCS SL confocal system, using a 100× oil immersion objective and Leica imaging software. GFP emission was captured between 500 and 550 nm and FM4-64 emission between 600 and 730 nm; excitation for both fluorophors was at 488 nm. Fluorographs shown are projection images generated from a single stack in the Z plane, with four-point line averaging.

Western blot analysis.

Procedures for Western blotting were performed essentially as described by Serrano et al. (30). The anti-σG polyclonal antiserum was incubated with membranes at a dilution of 1:1,000 in TBS-T (20 mM Tris-HCl [pH 7.6], 136 mM NaCl, 0.1% [vol/vol] Tween 20), containing 5.0% nonfat dry milk. Incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase was for 30 min at a 1:5,000 dilution, and detection was with an ECL Plus kit (Amersham). Protein samples of 300 μg were used in each lane.

Other methods.

β-Galactosidase was assayed essentially as described previously (23). Specific activity is expressed as nanomoles of ONPG (o-nitrophenyl-β-d-galactopyranoside) hydrolyzed per minute per milligram of bacterial dry weight; results of typical experiments are shown in the figures. B. subtilis transformation, sporulation by exhaustion in MSSM, and other methods were essentially as described previously (2, 38).

RESULTS

Deletion of the genes encoding σF and the anti-sigma factor SpoIIAB causes a breakdown of the tight progression of the activation of sporulation-specific σ factors.

When spoIIAC, the structural gene for σF, is inactivated by point mutation, no activity is detected for the later-expressed σ factors σE, σG, and σK (4, 13, 21). In contrast to that result, we have found that deletion of the entire spoIIA operon (designated spoIIAΔ) permits activation of σG (strain SL12436; Fig. Fig.3),3), although not σE or σK (not shown). Thus, σG becomes active in the absence of the activities of σF and σE, effectively disrupting the normal ordered activation of the sporulation-specific sigma factors. Activity was first apparent about 3 h after the onset of spore formation; no activity was detected in a strain with spoIIIG, the structural gene for σG, disrupted (SL11727; Fig. Fig.3).3). Similar results were obtained with a different σG-directed promoter (not shown). In the spoIIA deletion strain, activity was detected earlier during spore formation than for the corresponding spo+ strain (SL10369). However, although the normal tight regulation of σG had been disrupted in the spoIIAΔ mutant, no activity was detected during vegetative growth.

FIG. 3.
Activity of σG in a strain with the spoIIA locus deleted. The activity of σG is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains: filled squares, SL10369, spo+; open ...

The spoIIA operon encodes SpoIIAA and SpoIIAB, as well as σF; SpoIIAB is an anti-sigma factor for σF, and SpoIIAA is the anti-anti-sigma factor that interacts with SpoIIAB (reviewed in references 10 and 37). We tested to see whether deletion of spoIIAB and/or spoIIAA was necessary to obtain σG activity in the absence of σF. The σF-independent activation of σG was found to require deletion of spoIIAB (compare SL12434 with SL12432; Fig. Fig.4).4). SpoIIAB can act as an anti-sigma factor for σG, as well as for σF (5, 15, 32), so that its loss presumably permitted the establishment of a positive-feedback loop of σG-directed transcription of spoIIIG. Consistent with this interpretation, mutations in either spoIIIA or spoIIIJ, which ordinarily block σG activation in the mother cell through interaction with SpoIIAB (32), did not block σG activation in the spoIIA deletion background (data not shown).

FIG. 4.
The presence of SpoIIAB blocks σG activity in a strain that lacks σF. The activity of σG is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains: filled squares, SL12434, ...

The presence of spoIIAA reduced σG activity in the strain with spoIIAB and spoIIAC deleted (compare SL12432 with SL12436; Fig. Fig.4)4) but did not abolish it. SpoIIAA is known to inhibit activation of Spo0A (1), which is a central regulator of early sporulation gene expression (reviewed in reference 25). We think it plausible that inhibition of Spo0A activity by SpoIIAA accounts for the effects of SpoIIAA illustrated in Fig. Fig.4,4, for example, by reducing expression of the spoIIG operon (see below). However, we did not explore the role of SpoIIAA further.

The location of σG activation is switched from the prespore to the mother cell in spoIIA deletion mutants.

During normal spore formation, σG activity is confined to the prespore. The prespore specificity is established by the σF-directed transcription of spoIIIG, which is itself confined to the prespore. Once σG becomes active, a positive-feedback loop is then established in which σG directs spoIIIG transcription from the same promoter, which is recognized by both σF and σG (35, 36). The question arises, what happens in the absence of σF? To answer this, we monitored the expression of σG-directed sspA-gfp and spoIIIG-gfp transcriptional fusions. Consistent with extensive published results (reviewed in reference 10), their expression was largely confined to the prespore in a spo+ background (SL10969; Fig. Fig.55 and Table Table2).2). However, we have found that σG activity in spoIIAΔ strains was, within the limits of detection, confined to the mother cell in the majority of GFP-expressing organisms (Fig. (Fig.5,5, strains SL10034 and SL10153; Table Table2,2, SL10034, SL10153, and SL10162); back-crosses of the sspA-gfp fusion into a spo+ strain gave recombinants displaying prespore-specific expression, confirming that the fusion was unaltered. The mother cell location of σG activity in spoIIAΔ strains was surprising. However, it is consistent with recent results of Serrano et al. (32), who have found that SpoIIAB primarily regulates σG by preventing its activation in the mother cell, while having at most a redundant role in blocking σG activity in the prespore. That σG activity is detected after septation rather than completion of engulfment is probably also the result of loss of SpoIIAB control in the mother cell. The few cells that displayed whole-cell fluorescence (Table (Table2)2) did not contain a sporulation division septum.

FIG. 5.
Examples of GFP-expressing cells illustrating the patterns of localization of green fluorescence obtained with different strains containing a σG-directed sspA-gfp fusion. Bacteria were stained with FM4-64 to visualize membranes (red). A, SL10969 ...
TABLE 2.
Location of GFP expression from different gfp fusionsa

Read-through from the spoIIG locus is important for the mother cell location of σG expression.

A factor contributing to the mother cell location of σG activity in spoIIA deletion strains might be read through from the spoIIG locus, which is upstream of spoIIIG (Fig. (Fig.2).2). In spo+ strains spoIIIG is transcribed by read-through from the spoIIG locus, but there is no detectable translation of spoIIIG from this read-through transcript (20, 35). Indeed, relocating spoIIIG to the distal amyE locus does not impair σG activity, and gives efficient spore formation, so that read-through from spoIIG is not ordinarily required for spore formation (35). However, it may be that read-through is important for σG activation in the spoIIA deletion strains. Transcription from spoIIG through spoIIIG has been inferred primarily from results with transcriptional lacZ fusions (20, 35) and has proved difficult to detect reproducibly by Northern analysis or S1 mapping (references 16 and 20 and our unpublished observations). We have confirmed by reverse transcription-PCR that under sporulation conditions there was read-through of spoIIIG from spoIIGA in spoIIAΔ as well as in spo+ strains (data not shown).

To explore the role of this read-through, we tested the effect of relocating spoIIIG to amyE and found that spoIIIG was actively expressed in a spoIIAΔ strain (Fig. (Fig.6;6; SL11763). Thus, read-through from spoIIG was not necessary for expression of σG activity. However, the relocation changed the pattern of σG activity, as σG became active earlier in spore formation and became more active than when spoIIIG was at its natural locus (Fig. (Fig.6;6; SL12436). The reason for the earlier initiation of transcription of spoIIIG at the ectopic locus is not known. Importantly, in the great majority of organisms expressing the σG-directed sspA-gfp fusion, with spoIIIG located at amyE, the fluorescence was uncompartmentalized and the sporulation septum was not formed (Fig. (Fig.55 and Table Table2;2; SL11815). Thus, read-through of spoIIIG from spoIIG may be important for obtaining the mother cell specificity of σG activity observed in spoIIA deletion strains, even though it was not required to obtain the activity. It should be noted that Fujita and Losick (7) have reported greatly increased activity of the spoIIG promoter in the mother cell following septation in spo+ strains.

FIG. 6.
Effect on σG activity of relocating spoIIIG to an ectopic locus in a strain with the spoIIA locus deleted. The activity of σG is assessed as β-galactosidase activity expressed from an sspA-lacZ transcriptional fusion in the following ...

A second set of experiments reinforced the idea that read-through from spoIIG was indeed important for the mother cell specificity of σG expression and that the spoIIG promoter contributed to the strength of σG expression in the mother cell of spoIIAΔ strains. In these experiments, two spoIIA deletion strains were compared in which the spoIIG locus was also deleted but not spoIIIG. In one strain, the spoIIG promoter was retained so that it could potentially drive spoIIIG transcription, whereas in the other strain the promoter was not retained. The spoIIG region was replaced with the same cat cassette in the same orientation (away from spoIIIG) in both strains so that the insert should not cause a difference between the strains. There was substantial σG activity in the strain that retained the promoter (SL12137; Fig. Fig.7)7) and much-reduced activity in the strain that did not (SL12426; Fig. Fig.7);7); both strains displayed similar, abortively disporic phenotypes. In a strain that retained the spoIIG promoter, but not the spoIIG structural genes, σG activity was primarily confined to the mother cell (SL12306; Table Table2).2). Assessing the location of σG activity in a strain that lacked the promoter was problematic, as the activity was weak; in those cells that expressed sufficient GFP for an unambiguous determination, the activity was confined to the mother cell (SL12538; Table Table2).2). However, in other cells very weak GFP fluorescence was detectable at a level too low to permit determination of its location. Together, the results indicate that the spoIIG promoter contributed to strong mother-cell-specific σG activity in spoIIA deletion strains but that some mother-cell-specific activity could be obtained without that promoter.

FIG. 7.
Effect of deletions of the spoIIG operon on σG activity in strains with the spoIIA locus deleted. The activity of σG is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains with ...

Pro-σE and SpoIIGA control σG activity.

We detected no σE activity in the spoIIAΔ strains, consistent with previous results (14, 38) and indicating that σE was not needed for σG activity. However, inactivation of spoIIGB, which is the structural gene for pro-σE (12), blocked the appearance of σG activity in spoIIAΔ strains (Fig. (Fig.8;8; SL11758 without IPTG). Further, activity of σG was restored by expression of spoIIGB in trans from the IPTG-inducible Pspac(hy) promoter (Fig. (Fig.8;8; SL11758 with IPTG) so that the effect of spoIIGB inactivation on the appearance of σG activity cannot be explained by polarity on spoIIIG. Rather, the results suggest that either σE or pro-σE has a role in σG activation. Because no σE transcriptional activity was detected in spoIIAΔ strains, it seemed likely that pro-σE is required and not σE. Indeed, expression of a pro-less form of σE in a spoIIAΔ spoIIGBΔ mutant did not restore σG activity, although the strain did display σE activity (data not shown). These results suggest a previously unsuspected role for pro-σE that cannot be played by σE.

FIG. 8.
Effect of ectopic expression of spoIIGB on σG activity in a strain with the spoIIA locus deleted. The activity of σG is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains with ...

Expression of spoIIGB in trans resulted in much stronger and earlier σG activity in a spoIIAΔ strain (Fig. (Fig.8;8; SL11758 with IPTG) than when it was expressed in its natural position as part of the spoIIG locus (Fig. (Fig.8;8; SL12436). When spoIIGB was expressed in trans, the σG activity was uncompartmentalized, and no sporulation septa were formed (SL11813; Table Table22 and Fig. Fig.5).5). The lack of septa was consistent with the conclusion presented in the previous section that early activation of σG prevented spore septum formation. It remains to be established why σG became active earlier in SL11758. The spoIIGB gene was expressed earlier than when it was at its natural locus, as the inducer was present throughout growth and sporulation with strains SL11758 and SL12436; presumably, the early appearance of pro-σE somehow resulted in the early σG activity. Speculatively, pro-σE might interact with LonA or some other protease and so protect σG from proteolysis.

Pro-σE appears to be required only when SpoIIGA is produced. This conclusion is suggested by two sets of experiments. First, when both spoIIGA and spoIIGB were deleted, there was σG activity in a spoIIAΔ strain (SL12137; Fig. Fig.7),7), whereas when spoIIGB and not spoIIGA was deleted, no activity was detected (SL11758; Fig. Fig.8).8). Second, when both spoIIGA and spoIIGB were deleted, induction of spoIIGA in trans substantially reduced σG activity (compare SL12359 in the presence and absence of IPTG; Fig. Fig.9).9). The expression of σG activity in strain SL12359 even in the absence of IPTG was lower than in the corresponding strain, SL12137 (Fig. (Fig.7),7), that did not contain the Pspac(hy)-spoIIGA construct; we think that the reduced expression is a consequence of the leakiness of the inducible promoter. A clue to the possible role of pro-σE is provided by the observation that inactivation of lonA, which encodes an ATP-dependent protease (29), partly restored σG activity in a spoIIGB mutant strain (data not shown). LonA can degrade σG (29), and it may be that SpoIIGA sensitizes σG to proteolysis by LonA (or some other protease) and that somehow pro-σE but not σE can protect σG from the proteolysis. Our result is consistent with a role for SpoIIGA in facilitating LonA-directed proteolysis of σG, but it does not prove such a role.

FIG. 9.
Effect of ectopic expression of spoIIGA on σG activity in strains with the spoIIG and spoIIA loci deleted. The activity of σG is assessed as β-galactosidase expressed from an sspA-lacZ transcriptional fusion in the following strains ...

The loss of σG-directed transcriptional activity correlates with loss of the σG protein in an spoIIA deletion strain in which spoIIGB is also disrupted.

The loss of σG activity in spoIIAΔ strains with spoIIGB inactivated could result from absence of the σG protein or from the σG protein being held inactive. To distinguish between these possibilities, we used two approaches: first, immunoblotting with antibody directed against σG; second, fluorescence from a transcriptionally active σG-GFP fusion protein. The σG protein was first detected by immunoblotting 4 h after the end of exponential growth in spo+ and spoIIAΔ strains, and substantially more was detected by 6 h (strains SL10369 and SL12436, respectively; Fig. Fig.10).10). The presence of the σG protein correlated with σG activity as detected with an sspA-lacZ fusion (not shown). No σG protein was detected in an spoIIIG knockout mutant (strain SL11727). In contrast to the strong band observed for the spoIIAΔ mutant SL12436, the protein was barely detectable in a spoIIAΔ mutant with spoIIGB also inactivated (strain SL11671; Fig. Fig.10).10). This result indicated that inactivation of spoIIGB resulted in the almost total absence of the σG protein, not simply its inhibition, in the spoIIAΔ background. The presence of σG was not restored by expression in trans of a constitutively active form of σE in a strain with spoIIG deleted (strain SL12042; Fig. Fig.1010).

FIG. 10.
Effect of deletion of spoIIA and spoIIGB on the accumulation of σG during sporulation. Protein samples (300 μg) were obtained at the indicated time (h) after the start of spore formation in MSSM and fractionated by electrophoresis. They ...

We also utilized strains in which spoIIIG was replaced by a translational spoIIIG-gfp fusion via single-crossover (Campbell-like) plasmid integration. The fusion protein retained σG activity and did not block spore formation in a strain in which it was the sole copy of σG; the location of GFP is inferred to be a good indicator of the location of σG protein. In a spo+ strain, GFP fluorescence was located in the prespore (SL12673; Table Table2).2). When introduced into a spoIIAΔ strain, however, GFP fluorescence was confined to the mother cell (SL12674; Table Table2),2), correlating with the location of σG activity in spoIIAΔ strains. The result is consistent with mother-cell-specific spoIIIG transcription. No GFP was detected in a spoIIAΔ strain in which spoIIGB was also inactivated (data not shown), so there was no indication of σG being present in an inactive form in that strain.

σE activity in the mother cell does not block σG activity.

In strains deleted for spoIIA and with spoIIG intact, pro-σE is ordinarily not processed, and so σE is not active (38). We tested in two ways the effect on σG activity of having active σE in spoIIA deletion strains. (i) We introduced spoIIR under the control of the spoIIE promoter. spoIIR is the only σF-directed gene required for processing of pro-σE to its active form, and this construct results in σE activity in the absence of σF (38). Transcriptional activity of both σE and σG was detected with the construct (strains SL10215 and SL12518; Table Table2).2). As reported previously for σE (38), about half the GFP-expressing bacteria showed mother cell specificity; the rest showed whole-cell activity and had no sporulation septum, probably because the slightly earlier σE activation in that part of the population had prevented septum formation. Similar localization was observed for σG activity (SL10215; Table Table2).2). (ii) We inserted at thrC the gene for a constitutively active, pro-less form of σE. This construct resulted in lower σE activity than the PspoIIEspoIIR construct but a similar distribution of both σE activity and σG activity (not shown). Thus, as tested in two ways, σE did not have an antagonistic role towards σG. That many bacteria displayed mother-cell-specific σE activity reinforces the previous view that σF has at best a redundant role in directing σE activity to be confined to the mother cell (7, 38). The result with the pro-less form of σE suggests that processing of the pro sequence is not essential for compartmentalization of σE activity.

Both σE and σG were active before the completion of engulfment in strains SL10215 and SL12518. The σE activity in these strains enabled bacteria to complete engulfment (not shown); the corresponding strains, differing only by the lack of active σE, did not develop beyond septum formation. We infer that early activation of σG in the mother cell does not prevent engulfment. Both σE and σG activities were detected in the mother cell, suggesting no incompatibility between the two sigma factors, although we did not directly test whether they were active in the same mother cell. Presumably, both activities survive any competition with each other and with σA (18) for core RNA polymerase.

DISCUSSION

We report here that in the absence of both σF and the anti-sigma factor SpoIIAB, σG becomes active in the mother cell and not in the prespore during sporulation of B. subtilis. This is the first report, to our knowledge, of an efficient switch between prespore and mother cell of the location of activity of a sporulation-specific σ factor. The switch to mother cell location of the σG activity says that, at least in strains with spoIIAB and spoIIAC deleted, there is no “prespore-only” tag on σG and, likewise, no signal in the mother cell saying “no σG activity allowed.” SpoIIAB acts as an anti-sigma factor for σG as well as for σF (15, 17) and is thought to act against σG in vivo primarily to prevent inappropriate activation in the mother cell (32). Our results are consistent with this interpretation.

The other factor thought to contribute to the mother cell location of σG activity in strains with the spoIIA locus deleted is transcription of spoIIIG, the structural gene for σG, from upstream promoters, most notably the spoIIG promoter (Fig. (Fig.2).2). In support of this statement, relocating spoIIIG away from its normal location, which is downstream of the spoIIG locus, abolished the mother cell specificity. Also, mother-cell-specific σG activity was detected in a spoIIA deletion strain in which the spoIIG structural genes were deleted while leaving in place the spoIIG promoter upstream of spoIIIG (SL12306; Table Table2).2). Extending the deletion to include the spoIIG promoter substantially reduced σG activity (Fig. (Fig.7),7), indicating the importance of that promoter. However, residual mother-cell-specific σG activity remained even in the absence of the spoIIG promoter (SL12538; Table Table2),2), suggesting that some other promoter also played a role.

In Spo+ strains spoIIIG is transcribed productively (i.e., resulting in σG, which becomes active) from its own promoter. This transcription is primed by σF and so occurs only in the prespore. A positive-feedback loop is then established in which transcription is directed from the same promoter by σG (reviewed in reference 10). However, the spoIIIG locus is also transcribed by read-through from the spoIIG locus. The read-through transcript is normally translated poorly, if at all, probably because it forms a hairpin structure that sequesters the presumed ribosome binding site for spoIIIG (20, 35). Moving spoIIIG to an ectopic locus away from spoIIG does not impair spore formation in an otherwise spo+ strain, so any read-through transcript is clearly unnecessary for spore formation under the conditions used (35). Nevertheless, spoIIIG is located immediately downstream of spoIIG in all of the sequenced spore-forming bacteria (34). Such a juxtaposition suggests that in some circumstances the read-through may be important. Presumably in those circumstances the inhibitory effects of mRNA secondary structure can be overcome, as happens for the expression of rpoH and rpoS in E. coli (8), so as to produce some σG.

In strains with spoIIAB and spoIIAC deleted there is no σF priming and no SpoIIAB to block the activity of any σG formed in the mother cell as a result of read-through from spoIIG. In these circumstances, a small amount of active σG formed after the burst of spoIIG transcription that follows septation (7) may be sufficient to prime a positive-feedback loop of σG-directed transcription of spoIIIG. But now, the feedback loop is established in the mother cell, so that σG activity is confined to the mother cell. With respect to the prespore and the predivisional cell, expression from the spoIIG promoter is much reduced compared to that in the mother cell (7). Further, SpoIIAB has at most a redundant role in regulating σG in the prespore and also before septum formation, when other unidentified controls are thought to prevent activation (32). The net result is σG activity confined to the mother cell in strains with spoIIAB and spoIIAC deleted strains. Consistent with this interpretation, an spoIIIG-gfp translational fusion inserted at the spoIIIG locus is expressed only in the mother cell in a spoIIAΔ strain and only in the prespore in an spo+ strain (Table (Table22).

When spoIIIG was moved to an ectopic locus, amyE, away from the spoIIG promoter in spoIIAΔ strains, σG became active earlier during spore formation and was more active than when at its natural locus. It is not known why there was this earlier and stronger activity. Whatever the explanation, σG activity was uncompartmentalized and no sporulation septum was formed. As neither σF nor σE was active, the result suggests that σG activation can, like that of σE (11) and σF (3, 9), prevent subsequent septum formation. The function of such an inhibitory role for σG in a wild-type genetic background is not clear, but it may relate to the phenomenon of commitment, namely, the ability of an organism to continue to form a spore despite the addition of nutrients that might otherwise trigger an inappropriate restoration of growth and division (22). Thus, σG would prevent division of the prespore at later stages of spore formation when σF activity is thought to be curtailed (18).

We report a previously unrecognized control of σG activity involving pro-σE and SpoIIGA, which became apparent in spoIIAΔ strains. We found that in the presence of SpoIIGA, σG activity is only detected when pro-σE is also present. Two lines of evidence suggest that it is pro-σE and not σE that is required for this effect. First, no σE activity was detected in the spoIIA deletion strains that displayed σG activity; second, σG activity was not detected when a pro-less form of σE, and not pro-σE, was expressed from an ectopic locus, although σE activity was now detected. We think that pro-σE is needed for σG activation only when SpoIIGA is present, because σG activity was detected in strains with both spoIIGA and spoIIGB deleted. Presumably, pro-σE works to protect σG from protease action or from some other inhibitory mechanism that is stimulated by SpoIIGA. The amount of σG protein was dramatically reduced in the strain with spoIIGB deleted (Fig. (Fig.10),10), so we think it likely that the effect of pro-σE is to stabilize σG rather than to activate a preexisting inactive form.

The protease LonA has previously been shown to degrade σG (29, 31), and inactivation of lonA partly restored σG activity to a spoIIGBΔ spoIIAΔ mutant strain. It may be that LonA and SpoIIGA/pro-σE represent separate regulators of σG activity and that loss of LonA leads to a large σG increase that disrupts the other system. Alternatively, or additionally, pro-σE may protect σG from SpoIIGA acting to stimulate proteolysis of σG by LonA. The mechanism of SpoIIGA/pro-σE regulation remains unknown. Nevertheless, our results suggest that several partly overlapping mechanisms ordinarily act to prevent σG activation in the mother cell. They indicate that regulators of σE and σF can also regulate σG.

Acknowledgments

This work was supported by Public Health Service grant GM43577 (to P.J.P.) from the National Institutes of Health.

We thank Bettina Buttaro, Adriano Henriques, and Monica Serrano for helpful discussions. We thank Adriano Henriques and Monica Serrano for the antiserum to σG.

REFERENCES

1. Arabolaza, A. L., A. Nakamura, M. E. Pedrido, L. Martelotto, L. Orsario, and R. R. Grau. 2003. Characterization of a novel inhibitory feedback of the anti-anti-sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis. Mol. Microbiol. 47:1251-1263. [PubMed]
2. Chary, V. K., and P. J. Piggot. 2003. Postdivisional synthesis of the Sporosarcina ureae DNA translocase SpoIIIE either in the mother cell or in the prespore enables Bacillus subtilis to translocate DNA from the mother cell to the prespore. J. Bacteriol. 185:879-886. [PMC free article] [PubMed]
3. Coppolecchia, R., H. DeGrazia, and C. P. Moran, Jr. 1991. Deletion of spoIIAB blocks endospore formation in Bacillus subtilis at an early stage. J. Bacteriol. 173:6678-6685. [PMC free article] [PubMed]
4. Errington, J. 1993. Sporulation in Bacillus subtilis: regulation of gene expression and control of morphogenesis. Microbiol. Rev. 57:1-33. [PMC free article] [PubMed]
5. Evans, L., J. Clarkson, M. D. Yudkin, J. Errington, and A. Feucht. 2003. Analysis of the interaction between the transcription factor σG and the anti-sigma factor SpoIIAB of Bacillus subtilis. J. Bacteriol. 185:4615-4619. [PMC free article] [PubMed]
6. Francesconi, S. C., T. J. MacAlister, B. Setlow, and P. Setlow. 1988. Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis. J. Bacteriol. 170:5963-5967. [PMC free article] [PubMed]
7. Fujita, M., and R. Losick. 2002. An investigation into the compartmentalization of the sporulation transcription factor σE in Bacillus subtilis. Mol. Microbiol. 43:27-38. [PubMed]
8. Gruber, T. M., and C. A. Gross. 2003. Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol. 57:441-466. [PubMed]
9. Hilbert, D. W., and P. J. Piggot. 2003. Novel spoIIE mutation that causes uncompartmentalized σF activation in Bacillus subtilis. J. Bacteriol. 185:1590-1598. [PMC free article] [PubMed]
10. Hilbert, D. W., and P. J. Piggot. 2004. Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol. Mol. Biol. Rev. 68:234-262. [PMC free article] [PubMed]
11. Illing, N., and J. Errington. 1991. Genetic regulation of morphogenesis in Bacillus subtilis: roles of σE and σF in prespore engulfment. J. Bacteriol. 173:3159-3169. [PMC free article] [PubMed]
12. Jonas, R. M., E. A. Weaver, T. J. Kenney, C. P. Moran, Jr., and W. G. Haldenwang. 1988. The Bacillus subtilis spoIIG operon encodes both σE and a gene necessary for σE activation. J. Bacteriol. 170:507-511. [PMC free article] [PubMed]
13. Karmazyn-Campelli, C., C. Bonamy, B. Savelli, and P. Stragier. 1989. Tandem genes encoding σ-factors for consecutive steps of development in Bacillus subtilis. Genes Dev. 3:150-157. [PubMed]
14. Karow, M. L., P. Glaser, and P. J. Piggot. 1995. Identification of a gene, spoIIR, that links the activation of σE to the transcriptional activity of σF during sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 92:2012-2016. [PMC free article] [PubMed]
15. Kellner, E. M., A. Decatur, and C. P. Moran, Jr. 1996. Two-stage regulation of an anti-σ factor determines developmental fate during bacterial endospore formation. Mol. Microbiol. 21:913-924. [PubMed]
16. Kenny, T. J., and C. P. Moran, Jr. 1987. Organization and regulation of an operon that encodes a sporulation-essential σ factor in Bacillus subtilis. J. Bacteriol. 169:3329-3339. [PMC free article] [PubMed]
17. Kirchman, P. A., H. DeGrazia, E. M. Kellner, and C. P. Moran, Jr. 1993. Forespore-specific disappearance of the σ-factor antagonist SpoIIAB: implications for its role in determination of cell fate in Bacillus subtilis. Mol. Microbiol. 8:663-671. [PubMed]
18. Li, Z., and P. J. Piggot. 2001. Development of a two-part transcription probe to determine the completeness of temporal and spatial compartmentalization of gene expression during bacterial development. Proc. Natl. Acad. Sci. USA 98:12538-12543. [PMC free article] [PubMed]
19. Losick, R., and P. Stragier. 1992. Crisscross regulation of cell-type-specific gene expression during development in B. subtilis. Nature 355:601-604. [PubMed]
20. Masuda, E. S., H. Anaguchi, K. Yamada, and Y. Kobayashi. 1988. Two developmental genes encoding σ factor homologs are arranged in tandem in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 85:7637-7641. [PMC free article] [PubMed]
21. Partridge, S. R., and J. Errington. 1993. The importance of morphological events and intercellular interactions in the regulation of prespore-specific gene expression during sporulation in Bacillus subtilis. Mol. Microbiol. 8:945-955. [PubMed]
22. Piggot, P. J., and J. G. Coote. 1976. Genetic aspects of bacterial endospore formation. Bacteriol. Rev. 40:908-962. [PMC free article] [PubMed]
23. Piggot, P. J., and C. A. M. Curtis. 1987. Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions. J. Bacteriol. 169:1260-1266. [PMC free article] [PubMed]
24. Piggot, P. J., C. A. M. Curtis, and H. de Lencastre. 1984. Use of integrational plasmid vectors to determine the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. J. Gen. Microbiol. 130:2123-2136. [PubMed]
25. Piggot, P. J., and R. Losick. 2002. Sporulation genes and intercompartmental regulation, p. 483-518. In A. L. Sonenshein, J. A. Hoch and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington, D.C.
26. Pogliano, J., N. Osborne, M. D. Sharp, A. Abanes-De Mello, A. Perez, Y. L. Sun, and K. Pogliano. 1999. A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation. Mol. Microbiol. 31:1149-1159. [PMC free article] [PubMed]
27. Quisel, J. D., W. F. Burkholder, and A. Grossman. 2001. In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis. J. Bacteriol. 183:6573-6578. [PMC free article] [PubMed]
28. Schaeffer, P., J. Millet, and J. P. Aubert. 1965. Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 54:704-711. [PMC free article] [PubMed]
29. Schmidt, R., A. L. Decatur, P. N. Rather, C. P. Moran, Jr., and R. Losick. 1994. Bacillus subtilis Lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor σG. J. Bacteriol. 176:6528-6537. [PMC free article] [PubMed]
30. Serrano, M., L. Côrte, J. Opdyke, C. P. Moran, Jr., and A. O. Henriques. 2003. Expression of spoIIIJ in the prespore is sufficient for activation of σG and for sporulation of Bacillus subtilis. J. Bacteriol. 185:3905-3917. [PMC free article] [PubMed]
31. Serrano, M., S. Hövel, C. P. Moran, Jr., A. O. Henriques, and U. Völker. 2001. Forespore-specific transcription of the lonB gene during sporulation in Bacillus subtilis. J. Bacteriol. 183:2995-3003. [PMC free article] [PubMed]
32. Serrano, M., A. Neves, C. M. Soares, C. P. Moran, Jr., and A. O. Henriques. 2004. Role of the anti-sigma factor SpoIIAB in regulation of σG during sporulation of Bacillus subtilis. J. Bacteriol. 186:4000-4013. [PMC free article] [PubMed]
33. Stragier, P. 1989. Temporal and spatial control of gene expression during sporulation: from facts to speculations, p. 243-254. In I. Smith, R. A. Slepecky, and P. Setlow (ed.), Regulation of prokaryotic development. American Society for Microbiology, Washington, D.C.
34. Stragier, P. 2002. A gene odyssey: exploring the genomes of endospore-forming bacteria, p. 519-525. In A. L. Sonenshein, J. A. Hoch, and R. Losick (ed.), Bacillus subtilis and its closest relatives: from genes to cells. American Society for Microbiology, Washington DC.
35. Sun, D., R. M. Cabrera-Martinez, and P. Setlow. 1991. Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor σG. J. Bacteriol. 173:2977-2984. [PMC free article] [PubMed]
36. Sun, D., P. Stragier, and P. Setlow. 1989. Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis. Genes Dev. 3:141-149. [PubMed]
37. Yudkin, M. D., and J. Clarkson. 2005. Differential gene expression in genetically identical sister cells: the initiation of sporulation in Bacillus subtilis. Mol. Microbiol. 56:578-589. [PubMed]
38. Zhang, L., M. L. Higgins, P. J. Piggot, and M. L. Karow. 1996. Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis. J. Bacteriol. 178:2813-2817. [PMC free article] [PubMed]

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