An investigation into the compartmentalization of the sporulation transcription factor σE in Bacillus subtilis

© 2002 Blackwell Science Ltd

An investigation into the compartmentalization of thesporulation transcription factor sE in Bacillus subtilis

through the formation of a septum near one cell pole(Piggot and Coote, 1976). Asymmetric division gives riseto a small cell called the forespore and a large cell calledthe mother cell. Initially, the forespore and the mother celllie side by side. Later, however, the forespore is engulfedby, and wholly pinched off as a free protoplast within, themother cell, resulting in a sporangium that consists of acell within a cell. Differential gene expression commencesshortly after the formation of the polar septum, when thetranscription factors sE and sF direct gene transcription in the mother cell and the forespore respectively (Piggotand Losick, 2001). Here, we are concerned with the me-chanisms that restrict sE to the large chamber of the sporangium.

The sE factor is derived by regulated proteolysis fromthe inactive proprotein pro-sE, which has an N-terminalextension of 27 amino acids (LaBell et al., 1987; Miyaoet al., 1993). The conversion of pro-sE to mature sE

does not commence until after asymmetric division whenmature sE is largely confined to the mother cell. Propro-tein processing is dependent upon SpoIIGA, an integralmembrane protein and the putative pro-sE processingenzyme (Stragier et al., 1988; Peters and Haldenwang,1994). The genes for SpoIIGA (spoIIGA) and pro-sE

(spoIIGB) are contained within the two-cistron spoIIGoperon (Kenney and Moran, 1987; Jonas et al., 1988;Stragier et al., 1988). Transcription of spoIIG is inducedshortly after the onset of sporulation from a promoter thatis recognized by the phosphorylated form of Spo0A, themaster regulator for entry into sporulation acting in con-junction with RNA polymerase containing the house-keeping sigma factor sA (Satola et al., 1991; 1992).

SpoIIGA-mediated processing of pro-sE is controlled by an intercellular signal transduction pathway involvingthe secreted signalling protein SpoIIR (Hofmeister et al.,1995; Karow et al., 1995; Londono-Vallejo and Stragier,1995). SpoIIR is produced in the forespore under thecontrol of sF and is secreted, it is believed, into the spacebetween the two membranes of the polar septum, whereit interacts with the external domain of SpoIIGA, trig-gering the processing of pro-sE. The SpoIIGA–SpoIIRpathway is a timing device that ensures that processingdoes not commence until after sF is activated, which is inturn dependent upon the formation of the polar septum.The SpoIIGA–SpoIIR system is not, however, responsible(at least not by itself) for restricting sE-directed gene tran-scription to the mother cell. In cells engineered to produce

Molecular Microbiology (2002) 43(1), 27–38

Masaya Fujita and Richard Losick*Department of Molecular and Cellular Biology, HarvardUniversity, 16 Divinity Avenue, Cambridge, MA 02138,USA.

Summary

Sporulation in Bacillus subtilis involves the formationof a polar septum, which divides the sporangium intoa mother cell and a forespore. The sE factor, which isencoded within the spoIIG operon, is a cell-specificregulatory protein that directs gene transcription inthe mother cell. sE is synthesized as an inactive proprotein pro-sE, which is converted to the maturefactor by the putative processing enzyme SpoIIGA.Processing of pro-sE does not commence until afterasymmetric division when sE is largely confined tothe mother cell. Processing depends on the signallingprotein SpoIIR, which delays proteolysis until afterpolar septation, but the mechanism by which sE isconfined to the mother cell is not understood. Previ-ous work favoured a model in which pro-sE localizesto the mother cell face of the polar septum, such thatsE would be selectively released into mother cell cyto-plasm. Based on the use of green fluorescent protein(GFP) fusions, we now report that pro-sE is distributedapproximately uniformly along all membrane sur-faces and is not confined to the mothercell face ofthe septum. Rather, our results are consistent with a model in which preferential and persistent tran-scription of the spoIIG operon in the mother cell anddegradation of sE in the forespore contribute to the selective accumulation of sE in the mother cell.Persistent transcription of spoIIG after polar septa-tion also contributes to the proper timing of pro-sE

processing.

Introduction

One of the most fascinating challenges posed by differ-entiation in the spore-forming bacterium Bacillus subtilisis the question of how cell-specific gene expression isestablished. Shortly after the onset of spore formation, the developing cell or sporangium divides asymmetrically

Accepted 2 October, 2001. *For correspondence. E-mail [email protected]; Tel. (+1) 617 495 4905; Fax (+1) 617 496 4642.

SpoIIR before the formation of the polar septum, sE is frequently activated prematurely, but some sporangia areobserved in which sE-directed gene transcription is con-fined to the mother cell (Zhang et al., 1996).

What then is the nature of the mechanism(s) thatrestricts sE to the mother cell? Recent work based on theuse of a green fluorescent protein (GFP) fusion to the first55 amino acids of pro-sE (pro-sE55) support a model inwhich pro-sE localizes to the mother cell face of theseptum and, hence, mature sE is selectively released intothe mother cell cytoplasm after processing of the septum-associated proprotein (Ju and Haldenwang, 1999). Here,we report experiments based on the use of biologicallyactive fusions of GFP to full-length forms of the pro-sE

(pro-sE–GFP) and mature sE (sE–GFP) to investigate themechanisms by which sE is confined to the mother cell.We conclude that pro-sE is a membrane-associatedprotein, as reported previously (Hofmeister et al., 1995;Zhang et al., 1998), but that it is not confined to the mothercell face of the septum. Rather, it is distributed more orless uniformly along the cytoplasmic membrane and bothmembrane faces of the septum. We also observe that sE

continues to accumulate in the mother cell well after theformation of the polar septum. Based on the use of afusion of the gene for GFP to the promoter for the spoIIGoperon (PspoIIG–gfp), we conclude that this accumulationresults from selective and persistent transcription ofspoIIG in the mother cell. Finally, we present time-lapseimages consistent with the idea that sE–GFP is degradedin the forespore. In toto, our results are consistent with amodel in which multiple mechanisms contribute to thecompartmentalization of sE.

Results

A functional fusion of sE to GFP

We replaced the gene (spoIIGB) for pro-sE with a genefusion in which the coding sequence for GFP was fusedin frame to the 3¢ end of spoIIGB (see Experimental procedures). The resulting strain (MF56) sporulated with a frequency similar to that of the wild type, indicating that the sE–GFP fusion protein was partially if not fullyfunctional. Consistent with the sporulation results, pro-sE–GFP was efficiently processed to sE–GFP in aspoIIGA-dependent manner (Fig. 1).

Localization of pro-sE–GFP and sE–GFPduring sporulation

We examined the localization of the GFP fusion proteinsby fluorescence microscopy. Because microscopy couldnot distinguish between the proprotein and mature formsof the fusion protein, we refer to the fluorescence as

simply indicating the localization of pro-sE–GFP/sE–GFP(column 1 in Fig. 2) but, in cases of membrane localiza-tion, we presume that the signal is largely or exclusivelycaused by pro-sE–GFP and, in the cases of cytoplasmiclocalization, we presume that the signal is largely causedby sE–GFP. The cells were also stained with the vitalmembrane stain FM4-64 (red; Fig. 2, column 3) and withthe DNA stain DAPI (blue; Fig. 2, column 5).

In sporangia that had not yet reached the stage ofasymmetric division, a low-level fluorescence from pro-sE–GFP/sE–GFP was observed at the cytoplasmicmembrane (Fig. 2A). Later, after asymmetric division, astrong signal from GFP was observed at the polar septum (Fig. 2B). At first glance, it appears that pro-sE–GFP/sE–GFP (which we interpreted as representingpro-sE) is enriched at the polar septum, and similar resultshave been interpreted thus previously (Hofmeister, 1998;Ju and Haldenwang, 1999). But we note that membranestaining with FM4-64 gave a similar higher intensity at the polar septum than at the cytoplasmic membrane. Astraightforward explanation for the strong signal is that theseptum consists of two adjacent layers of membrane, onefrom the forespore and one from the mother cell (Poglianoet al., 1999). Thus, the double membranes could con-tribute to the stronger signal. Also, the septum is a disk,and visualizing it edgewise could lead to a brighter signalthan at the cell periphery. The important point is that theGFP signalling appeared to be no more enriched at the septum than the FM4-64 signal. Indeed, quantitative

© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 27–38

28 M. Fujita and R. Losick

Fig. 1. Processing of pro-sE–GFP during sporulation. Whole-cellextracts from cells at hour 2.5 of sporulation were loaded on a 12%SDS–polyacrylamide gel and analysed by immunoblotting usinganti-GFP antibodies. Samples were prepared from MF56(spoIIGB–gfp; lane 1) and from MF75 (spoIIGAD17 spoIIGB–gfp;lane2). The sizes of protein markers (kDa) are indicated on the left.

Compartmentalization of sE in B. subtilis 29

experiments (data not shown) in which we scannedacross sporangia indicated no more enrichment (within avariance of about 10%) for GFP than for FM4-64. By thisreasoning, we conclude that pro-sE may not be enrichedat the septum. Rather, it is uniformly distributed along allmembrane surfaces in the sporangium, whether cyto-plasmic or septal, and only appears to be enriched at theseptum because of the two membrane layers and theeffect of looking at the septum edgewise.

At later times, when the process of engulfment hadcommenced (as judged by the arching of the septal mem-branes around the forespore in Fig. 2C and D), a markedchange in the pattern of fluorescence was observed.Rather than being restricted to the cell periphery and theseptum, fluorescence was observed in the cytoplasm ofthe mother cell but was absent from the forespore. Insome sporangia (e.g. see Fig. 2D), the fluorescenceseemed to co-localize with the mother cell nucleoid. Weinterpret these results to indicate that pro-sE is convertedto sE at or just before the process of engulfment com-mences, at which time the mature form of the transcrip-tion factor is released into the cytoplasm, where theresulting complex of RNA polymerase with sE binds to andinitiates transcription from the mother cell chromosome. Asimilar pattern of mother cell fluorescence was observedat still later times, when the process of engulfment was

nearing completion (as judged by the presence of a com-plete circle of FM4-64 staining around the forespore inFig. 2E) or had reached completion (Fig. 2F). (That thesporangia in Fig. 2F had completed engulfment is indi-cated by the absence of forespore staining; because it is unable to penetrate across a membrane, FM4-64 fails to stain forespores that have been fully pinched off as a free protoplast within the mother cell; Sun et al.,2000.)

Use of protoplasts to visualize the distribution of pro-sE–GFP/sE–GFP between the forespore and the mother cell

Ju and Haldenwang (1999) have reported that pro-sE

localizes to the mother cell face of the septum based on the use of a fusion of the first 55 residues of the pro-protein to GFP and experiments in which sporulating cells harbouring the fusion were treated with lysozyme.Lysozyme removes the outer layer of peptidoglycan fromthe sporangium and the peptidoglycan layer in the polarseptum, thereby converting the sporangium into twoattached protoplasts: a large one corresponding to themother cell and a small one corresponding to the fores-pore (Wu et al., 1998; Ju and Haldenwang, 1999; Kinget al., 1999). Because the resulting protoplasts lack a


Fig. 2. Subcellular localization of pro-sE/sE–GFP during sporulation. A straincarrying the spoIIGB–gfp fusion (MF56) wasinduced to sporulate, and cells wereexamined by fluorescence microscopy. Thesporangia were treated with the vitalmembrane stain FM4-64 and with the DNAstain DAPI to visualize membranes and DNArespectively. Column 1, fluorescence frompro-sE/sE–GFP; column 2, merged images offluorescence from FM4-64 and from pro-sE/sE–GFP; column 3, fluorescence from FM4-64; column 4, overlay offluorescence from FM4-64 and from DAPI;column 5, fluorescence from DAPI.A. Sporangia at the predivisional stage.B. Sporangia immediately after polarseptation.C. Sporangia in which the mother cellmembrane has started to move up andaround the forespore.D. Sporangia at a more advanced stage ofengulfment.E. Sporangia in which the engulfingmembranes have encircled the forespore.F. Sporangia in which forespore is fullypinched off as a free protoplast within themother cell and hence is unstained with FM4-64.The identical field of cells is shown acrosseach row. The scale bar in (A) is one micron.

septum, the distribution of pro-sE–GFP/sE–GFP betweenthe forespore and the mother cell can be inferred directlyfrom the relative fluorescence of the two protoplasts (Ju and Haldenwang, 1999). [Because the pro-sE55–GFPfusion was non-functional and because forespore com-partments are generated at both poles of the sporangiumin the absence of sE activity (Piggot and Coote, 1976), the lysozyme-treated sporangia of Ju and Haldenwang(1999) consisted of two forespore protoplasts and onemother cell protoplast.]

We repeated these experiments using our functionalpro-sE–GFP fusion protein, collecting samples at inter-vals after the start of sporulation, treating the sporangia with lysozyme and examining the treated sporangia by fluorescence microscopy. We observed that, at earlytimes after asymmetric division, fluorescence was dis-tributed approximately equally between the forespore andmother cell protoplasts for most of the treated sporangia in the field (an example is shown in Fig. 3A).At later times, however, the distribution of fluorescencewas strongly biased to the mother cell protoplast for many of the sporangia, and the overall strength of fluo-rescence signal was much greater than at earlier times(an example is shown in Fig. 3B). We conclude that pro-sE is present in approximately equal concentrations in theforespore and the mother cell at the time of polar divisionbut over time accumulates to high levels in the mothercell. The simplest interpretation of these results is thatsynthesis of pro-sE continues after asymmetric divisionand that this synthesis occurs selectively in the mothercell or that pro-sE (or sE) is degraded in the forespore or both.

Preferential transcription of the gene for pro-sE in themother cell

To investigate whether the gene for pro-sE is preferentiallytranscribed in the mother cell, we created fusions of the promoter for the spoIIG operon to gfp (PspoIIG–gfp) and to lacZ (PspoIIG–lacZ). As shown in the time course experiment in Fig. 4A and B, green fluorescence fromPspoIIG–gfp continued to accumulate selectively in themother cell during and after engulfment, the fluorescentsignal becoming quite intense in the mother cell after the completion of engulfment (when, as seen in the hourthree column, and owing to the lipophilic nature of FM4-64, the dye is unable to stain forespores that are fully engulfed). As a reference, Fig. 4C and D show thecorresponding pattern of accumulation of pro-sE–GFP/sE–GFP. As a control for the results with the PspoIIG–gfpfusion, Fig. 4E and F show that GFP from a fusion of gfp to the Pspac

C promoter accumulated much more uniformly throughout the sporangium and was not re-stricted to the mother cell. Pspac

C is a modified form of the IPTG-inducible Pspac promoter that lacks the operatorfor the LacI repressor and hence is active constitutively(see the Experimental procedures). This result indicatesthat the selective accumulation of GFP in the mother cellthat was observed with the PspoIIG–gfp-containing strainwas not the result of instability of GFP in the foresporebut rather of a difference in transcription between the two compartments.

Finally, as further indication that the spoIIG operon continues to be expressed after polar division, the time course in Fig. 5 shows that the accumulation of b-galactosidase from the PspoIIG–lacZ fusion was largelycoincident with, or subsequent to, the appearance of spo-rangia that had undergone asymmetric division.

Effect of switching the promoter for spoIIG on the compartmentalization of sE

Next, we investigated the effect of switching the promoterfor the spoIIG operon from PspoIIG to Pspac

C on the com-partmentalization of sE. Using a strain (MF463) harbour-ing a construct (Pspac

C–spoIIGAB–gfp) in which gfp wasfused to the gene (spoIIGB) for pro-sE and in which thePspoIIG promoter was replaced with the Pspac

C promoter, weexamined the distribution of pro-sE–GFP/sE–GFP by flu-orescence microscopy in intact sporangia and in sporan-gia that had been treated with lysozyme. We frequentlyobserved sporangia at the stage of polar division, in whichfluorescence was more or less uniformly distributedthroughout the membranes or the cytoplasm of the spo-rangium (Fig. 6A). Among sporangia that had reached thestage of engulfment, however, fluorescence was gener-ally restricted to the mother cell (indicated by arrows in



Fig. 3. Distribution of pro-sE/sE–GFP between mother cell andforespore protoplasts. Protoplasts were generated by lysozymetreatment of sporangia from the pro-sE/sE–GFP-producing strainMF56. Fluorescence microscopy was used to visualizefluorescence from pro-sE/sE–GFP in column 1 or from DAPI incolumn 3. Lane 2 shows merged images of fluorescence from GFPand DAPI. Lane 4 shows phase-contrast images of the protoplasts.A. Protoplasts of a sporangium from cells collected at the stage ofpolar septation. The scale bar is one micron.B. Protoplasts from a sporangium from cells at the stage ofengulfment.


Fig. 6A). When the sporangia were treated with lysozyme,we observed three patterns of fluorescence: sporangia inwhich fluorescence (presumably from pro-sE–GFP) wasassociated with the membrane of both the mother cell andthe forespore protoplast (Fig. 6C); sporangia in which fluorescence (presumably from sE–GFP) was presentthroughout the mother cell protoplast but restricted to themembrane of the forespore protoplast (Fig. 6D); and spo-

rangia in which fluorescence was present throughout boththe mother cell and forespore protoplasts (Fig. 6E). Weconclude that use of the Pspac

C promoter was sufficientpartially to break the compartmentalization of pro-sE–GFPand sE–GFP, at least at early times after the formation ofthe polar septum.

Nevertheless, use of PspacC did not measurably alter the

compartmentalization of sE-directed gene transcription.Using a strain (MF416) harbouring the Pspac

C–spoIIGfusion, we found that sE-directed synthesis of GFP (froma PspoIID–gfp fusion) was largely restricted to the mothercell (Fig. 6B and Table 1). Also, the efficiency of sporula-tion of strains MF463 and MF416 was indistinguishablefrom the wild type (ª 2 ¥ 108 spores ml–1). The obser-vation that sE–GFP and sE activity were restricted to the mother cell compartment even though they wereexpressed uniformly under the control of the Pspac

C pro-moter suggested that additional mechanisms contribute tothe compartmentalization of sE.

Degradation of pro-sE–GFP/sE–GFP in the forespore

If selective and persistent transcription of spoIIG in themother cell is insufficient to explain the compartmental-ization of sE-directed transcription, then what additional


Fig. 4. Distribution of GFP generated from thepromoter for the spoIIG operon.A. GFP localization in MF237(amyE::PspoIIG–gfp).B. FM4-64 staining of MF237.C. Pro-sE/sE–GFP localization in MF56(spoIIGB–gfp).D. FM4-64 staining of MF56.E. GFP localization in MF339(amyE::Pspac

C–gfp).F. FM4-64 staining of MF339. Cells wereexamined at the indicated times after the startof sporulation.

Fig. 5. Time course of accumulation of b-galactosidase fromPspoIIG–lacZ. Culture samples from strain MF290 were collected at 0.5 h intervals after the start of sporulation and analysed for b-galactosidase (closed squares) and for polar septation (shadedbars). Cells with polar septa were assessed both by FM4-64 andDAPI staining.

mechanism(s) contributes to restricting the action of sE tothe large chamber of the sporangium? When pro-sE–GFPwas produced under the control of the Pspac

C promoter, flu-orescence was frequently observed throughout the spo-rangium in sporangia that had undergone polar division,but became restricted to the mother cell by the time ofengulfment. A simple interpretation of this observation isthat pro-sE–GFP or sE–GFP is degraded in the forespore.To investigate this possibility, we looked at the fate of pro-sE–GFP/sE–GFP by time-lapse microscopy. Cells that hadbeen collected at 90 min after the start of sporulation wereapplied to an agarose bed and viewed at 10 min intervals.

The time-lapse images in Fig. 7A show two sporangia in which fluorescence was initially distributed throughoutthe sporangium. Our interpretation of these sporangia is that processing had occurred, and mature sE–GFPwas initially present in both the forespore and the mo-ther cell. Over time, however, the fluorescent signal selec-tively and progressively disappeared from the foresporecompartment.

We interpret these results to indicate that sE is de-graded in the forespore but, strictly speaking, we could be observing the loss of only the GFP moiety of thesE–GFP fusion protein in the forespore. It is likely, how-



Fig. 6. Effect of the PspacC promoter on the

localization of pro-sE/sE–GFP.A. Localization of pro-sE/sE–GFP in (column1) and FM4-64 staining of (column 3)sporangia from strain MF463 (spoIIGABD::tetamyE::Pspac

C–spoIIGAB-gfp).B. Localization of GFP in (column 1) andFM4-64 staining of (column 3) sporangia fromstrain MF416 (spoIIGABD::tetamyE::Pspac

C–spoIIGAB, thrC::PspoIID–gfp).Column 2 shows overlays of columns 1 and3. The arrows point to forespores. Insporangia that had completed engulfment, noFM4-64 staining of the forespore is observedbecause the forespore is pinched off as a freeprotoplast (Pogliano et al., 1999).C–E. Three patterns of pro-sE/sE–GFPlocalization in protoplast cells from MF463.Column 1, fluorescence from pro-sE/sE–GFP;column 2, overlay of GFP and DAPI staining;column 3, fluorescence from DAPI staining;column 4, phase-contrast microscopy. Cellswere analysed at hour two of sporulation.


ever, that the loss of fluorescence reflects the degrada-tion of the entire pro-sE–GFP fusion protein because littleor no loss of fluorescence in the forespore was observedin the experiments in Fig. 4E, in which GFP alone wasproduced under the control of Pspac

C. An alternative inter-pretation is that sE–GFP is degraded equally in both com-partments but is only replenished in the mother cell as aresult of selective transcription. We think that this isunlikely based on the observation (Fig. 6A) that uniformexpression of spoIIGB–gfp under the control of Pspac

C

resulted in compartmentalized sE–GFP.[We note in Table 1 that the proportion of sporangia

exhibiting compartmentalized sE activity (as monitored by the fluorescence of PspoIID–gfp in cells harbouring a Pspac

C–spoIIG fusion) was higher than the proportionexhibiting compartmentalized sE–GFP protein (as moni-tored by fluorescence from Pspac

C–spoIIGAB–gfp). If, aswe suggest, sE–GFP undergoes proteolysis in the fores-

pore, then it is possible that proteolytic intermediates inthe degradation of sE–GFP are capable of continued flu-orescence even though they are incapable of directingtranscription. Alternatively, the results in Table 1 couldindicate the existence of an inhibitor of sE activity in theforespore. Finally, the discrepancy between sE activityand sE–GFP could be more apparent than real, in that itwas difficult to distinguish a low level of fluorescence inthe forespore from no fluorescence in the forespore withthe Pspac

C–spoIIGAB–gfp fusion, and we tended to scoreeven a weak signal as positive.]

Not all the sporangia we observed conformed to thepattern represented in Fig. 7A. The time-lapse images inFig. 7B, for example, show a sporangium at the stage ofpolar division in which the fluorescent signal is largelycoincident with the cytoplasmic and septal membranes at the earliest time point. Over time, the signal spreads to the cytoplasm in the mother cell, which we interpret


Table 1. Localization of sE–GFP and of sE activity in cells harbouring PspacC–spoIIG.

Percentage of sporangia with the indicated pattern of GFP localization

Hours after the TotalStrain GFP fusion onset of sporulation sporangia MCa FSb Both

MF416c PspoIID–gfp 2.5 128 94 0 63.0 135 97 0 33.5 91 95 0 5

MF463d spoIIGB–gfpe 2.5 49 52 0 483.0 69 71 0 293.5 93 77 0 23

a. Localization in the mother cell only.b. Localization in the forespore only.c. MF416 (spoIIGABD::tet, amyE::Pspac

C–spoIIGAB spc, thrC::PspoIID–gfp erm).d. MF463 (spoIIGABD::tet, amyE::Pspac

C–spoIIGAB–gfp spc kan).e. sE–GFP that had been processed was scored.

Fig. 7. Time-lapse microscopy of fluorescence from pro-sE/sE–GFP generated from the constitutive promoter PspacC. MF463 cells

(spoIIGABD::tet, amyE::PspacC–spoIIGAB–gfp) were monitored commencing at 90 min after the start of sporulation. Time-lapse intervals are

indicated in minutes from the first observation time.A. The disappearance of sE in the forespore is indicated by arrows.B. The transcription factor is initially in the membrane-associated proprotein form and is then processed to sE–GFP when fluorescence fromthe fusion protein can be detected more strongly in the mother cell cytoplasm than in the forespore cytoplasm. The bright dots are artifacts ofthe fluorescence microscopy.

as reflecting the conversion of pro-sE–GFP to maturesE–GFP. In the forespore, however, the signal remainedassociated with the membrane, and little fluorescencewas observed in the cytoplasm. Evidently, processing wasnot occurring in the forespore chamber of this sporangiumor, if it was occurring, the resulting mature sE–GFP wasdegraded too rapidly for us to observe by time-lapsemicroscopy.

Effect of SpoIIR on the timing of pro-sE processing

Finally, we investigated the role of the signalling proteinSpoIIR in the timing of pro-sE processing. SpoIIR is asecreted signalling protein that is produced in the fores-pore under the control of sF. To uncouple the timing ofSpoIIR production from its role in the activation of pro-sE,we constructed a strain (MF444) in which the spoIIGoperon was under the control of constitutive promoterPspac

C and contained two copies of spoIIR: one copy thatwas under its normal promoter and one that was underthe control of the IPTG-inducible promoter Pspac

IN. Forcomparison, we constructed a strain (MF135) in which thespoIIG operon was under the control of its normal pro-moter and that contained a single copy of spoIIR underthe control of Pspac

IN. Western blot analysis showed that,when MF135 cells were grown in the absence of IPTG,the accumulation of pro-sE commenced at a similar time to that observed in the wild type, but little processing was observed (compare Fig. 8A with Fig. 8B). Also, asexpected, the level of sporulation was low. When theMF135 cells were grown in the presence of inducer,however, processing of pro-sE could be observed, andthis processing commenced at a similar time to that

observed in the wild type (Fig. 8C). As a result, the cellssporulated relatively efficiently (Fig. 8C). As shown above,pro-sE from the spoIIG promoter largely accumulates afterseptation, which evidently explains why the timing of pro-sE processing in MF135 was similar to that observed inthe wild type, even though SpoIIR was produced in thepredivisional sporangium.

Finally, when cells of MF444 were grown in the pres-ence of inducer, processing of pro-sE commenced pre-maturely, and only a low level of sporulation was observed(Fig. 8E). Fluorescence microscopy (data not shown)revealed that the cells were blocked at the predivisionalstage of sporulation. As a control, when the cells weregrown in the absence IPTG (conditions under whichspoIIR would be expressed exclusively from its normalpromoter), processing commenced at a similar time tothat of the wild type, and a high level of sporulation wasobserved (Fig. 8D).

We conclude that the timing of pro-sE activation is crit-ical for efficient sporulation and that the timing of pro-sE

synthesis and SpoIIR synthesis is a partially redundantmechanism that ensures that mature sE does not appearuntil about the time of asymmetric division. Under condi-tions in which transcription of either the spoIIG operon orthe spoIIR gene was driven from its normal promoter, a relatively high level of sporulation was observed.However, when both spoIIG and spoIIR were transcribedfrom the spac promoter, sporulation was impaired.

Discussion

We have presented evidence that transcription of thespoIIG operon persists after polar division and that this



Fig. 8. Effect of SpoIIR on the timing of pro-sE processing. Whole-cell extracts from sporulating cells were loaded on a 12%SDS–polyacrylamide gel and analysed by immunoblotting using anti-sE antibodies. Samples were prepared from: (A) PY79; (B) MF135(spoIIRD::km, thrC::Pspac

IN–spoIIR) without IPTG; (C) MF135 that had been grown in the presence of 1 mM IPTG; (D) MF444 (spoIIGABD::tet,amyE::Pspac

C–spoIIGAB, thrC::PspacIN–spoIIR) without IPTG; (E) MF444 that had been grown with 1 mM IPTG. Samples were taken at the times

indicated after the start of sporulation. The positions of pro-sE and sE are indicated on the right-hand side of the gel. The right-hand columnshows the frequency of heat-resistant spore formation for the cells in (B–E) expressed as a fraction of that observed for the wild type (A).


transcription occurs preferentially in the mother cell. Evi-dence for continued transcription of spoIIG in the mothercell came from experiments in which we observed con-tinued accumulation of GFP in the mother cell from aPspoIIG–gfp fusion well after the time of asymmetric divi-sion. As a control, the Pspac

C promoter directed the accu-mulation of GFP uniformly throughout the sporangium.We do not know the basis for differential transcription fromthe PspoIIG promoter, but it is conceivable that Spo0A actspreferentially in the mother cell. Both PspoIIG and Pspac

C aretranscribed by sA-containing RNA polymerase, but tran-scription from PspoIIG is additionally dependent upon the phosphorylated form (Spo0A~P) of the DNA-bindingprotein Spo0A. Perhaps Spo0A accumulates preferen-tially or is somehow preferentially phosphorylated in thelarge chamber of the sporangium.

Preferential and persistent transcription from PspoIIG inthe mother cell could contribute to the compartmentaliza-tion of sE. However, if so, its contribution is masked by asecond pathway that is responsible for limiting sE to themother cell. This conclusion rests on experiments in whichthe promoter (PspoIIG) for the spoIIG operon was replacedby the spac (Pspac

C) promoter. Cells harbouring thePspac

C–spoIIG construct sporulated at a normal efficiencyand were not measurably impaired in restricting sE-directed gene transcription to the mother cell.

A second and perhaps more important pathway con-tributing to the compartmentalization of the mother celltranscription factor could be selective degradation of pro-sE or sE or both in the forespore. Our evidence for this isbased on the use of a Pspac

C–spoIIG–gfp construct. Insporulating cells harbouring this construct, fluorescencefrom the fusion protein initially filled the entire spo-rangium. Over time, however, fluorescence was selec-tively eliminated from the forespore, and we succeededin visualizing this disappearance by time-lapse micro-scopy. Strictly speaking, our experiments reveal the lossof GFP, not of sE itself. However, GFP produced underthe control of Pspac

C did not disappear from the forespore.The simplest interpretation of our results is that GFP wasbeing degraded as a consequence of its being fused tosE and that sE (and sE–GFP) is subject to proteolysis inthe forespore. Because the evidence for degradation ofsE is indirect (degradation of GFP), we consider the con-clusion that sE is eliminated from the forespore by prote-olysis to be provisional. Nevertheless, degradation in theforespore could be the principal mechanism by which sE

is restricted to the forespore.Post-septational transcription of the spoIIG operon also

appears to contribute to the proper timing of pro-sE pro-cessing. It is known that the timing of pro-sE processingis governed in part by the SpoIIR–SpoIIGA pathway,which delays proteolysis until after septation (Hofmeisteret al., 1995; Zhang et al., 1996; Pogliano et al., 1997).

However, cells in which the normal timing function of theSpoIIGA–SpoIIR pathway was disrupted through prema-ture synthesis of SpoIIR were only modestly impaired insporulation. Thus, as noted previously, delaying the syn-thesis of SpoIIR until after polar septation is not essentialfor sporulation or for the compartmentalization of sE-directed gene expression (Zhang et al., 1996). However,cells that both harboured the Pspac

C–spoIIG construct andin which SpoIIR synthesis was induced prematurelyexhibited a pronounced defect in the efficiency of sporeformation. We conclude that delaying the synthesis ofSpoIIR until after asymmetric division and partially delay-ing transcription of spoIIG until after septation are partiallyredundant pathways for achieving proper temporal controlof sE-directed transcription. Only when both pathways areimpaired is sporulation markedly impaired.

Previous models for restricting sF- and sE-directed geneexpression to the forespore and mother cell, respectively,were based on the concept of sequestration to one orother face of the polar septum. For example, in the caseof sF, Wu et al. (1998) claimed that a key protein in thepathway that controls the activation of sF, the phos-phatase SpoIIE, which is known to localize to the polarseptum, is sequestered on the forespore face of theseptum, thereby restricting sF activation to the forespore.Meanwhile, Ju and Haldenwang (1999) have presentedevidence indicating that pro-sE localizes to the mother cellface of the septum, thereby explaining how mature sE

comes to localize to the mother cell. Wu et al. (1998)treated sporulating cells with lysozyme to convert the fore-spore and the mother cell into protoplasts, which releasedSpoIIE–GFP from the septum. They observed greater flu-orescence in the forespore protoplast than in the mothercell protoplast, which they interpreted as indicating thatSpoIIE–GFP was restricted to the forespore face of theseptum. However, because the forespore is much smallerthan the mother cell, the intensity of fluorescence in theforespore would be expected to be greater than that in themother cell even if SpoIIE–GFP were localized equally toboth faces of the septum (King et al., 1999). In the caseof pro-sE, earlier work indicated that the proprotein local-izes to the septum (Ju et al., 1997; Hofmeister, 1998), andthe recent work by Ju and Haldenwang (1999), onceagain based on the use of protoplasts, indicates that pro-sE is sequestered to the mother cell face of the septum.However, we find that the distribution of pro-sE–GFPclosely matches the distribution of membrane, as judgedby use of the vital membrane stain FM4-64, and that the apparent enrichment of pro-sE–GFP at the septum is sufficiently explained by the fact that the septum is composed of membranes and is being viewed in an edge-wise manner. Moreover, our current finding of persistenttranscription of the spoIIG operon in the mother cell pro-vides a sufficient explanation for the strong GFP signal


observed in the mother cell protoplast. If proteins localizein an asymmetric manner to one or other face of thesporulation septum, clear evidence for such a situation isnot yet available.

In summary, we investigated mechanisms involved inthe compartmentalization of sE and the proper timing ofpro-sE processing. Our evidence does not support a pre-viously proposed model for compartmentalization basedon the sequestration of pro-sE to the mother cell face of the septum. Instead, our results suggest that persistentand preferential transcription of the spoIIG operon in themother cell and preferential degradation of sE in the forespore contribute to the compartmentalization of themother cell transcription factor, with degradation being the principal mechanism for restricting sE to one cell.Post-divisional transcription of spoIIG may additionallycontribute to the proper timing of pro-sE processing but is redundant with the SpoIIGA–SpoIIR pathway, whichdelays processing until after septation. Multiple over-lapping pathways may help to ensure tight temporal and spatial control of the mother cell transcription factor.

Experimental procedures

Strains

All strains used in this study were derivatives of the wild-typeB. subtilis strain PY79 (Youngman et al., 1984). Strain MF56(spoIIGB–gfp spc) was constructed by integration of pMF7(see below) into the chromosome at the spoIIGB locus of PY79. Strain MF75 (spoIIGAD17, spoIIGB–gfp spc) wasconstructed by transformation of RL879 (spoIIGAD17, labora-tory stock) with chromosomal DNA from MF56. StrainsMF237 (amyE::PspoIIG–gfp spc), MF339 (amyE::Pspac

C–gfpspc), MF382 (amyE::Pspac

C–spoIIGAB spc), MF290(amyE::PspoIIG–lacZ spc) and MF160 (thrC::PspoIID–gfp erm)were constructed by double recombination at amyE or thrCusing plasmids (see below) pMF19, pMF35, pMF46, pMF27and pMF15 respectively.

MF135 (spoIIRD::kan, thrC::PspacIN–spoIIR erm) was

constructed by transformation of RL1965 (spoIIRD::kan, laboratory stock) with chromosomal DNA from RL1966(thrC::Pspac

IN–spoIIR erm, laboratory stock). Strain MF390(spoIIGABD::tet, amyE::Pspac

C–spoIIGAB spc) was con-structed by transformation of MF382 (amyE::Pspac

C–spoIIGABspc) with chromosomal DNA from MO1780 (spoIIGABD::tet,trpC2, pheA1), a gift from P. Stragier (Institut de BiologiePhysico-Chimique, Paris, France). Strain MF444 (spoI-IGABD::tet, amyE::Pspac

C–spoIIGAB spc, thrC::PspacIN–spoIIR

erm) was constructed by transformation of MF390 with chromosomal DNA from RL1966 (thrC::Pspac

IN–spoIIR erm, laboratory stock). Strain MF463 (spoIIGABD::tet,amyE::Pspac

C–spoIIGAB–gfp spc kan) was constructed bysingle cross-over recombination of plasmid pMF6 (seebelow) at the amyE::Pspac

C–spoIIGAB spc locus of strainMF390. Strain MF416 (spoIIGABD::tet, amyE::Pspac

C–spoI-

IGAB spc, thrC::PspoIID–gfp erm) was constructed by transfor-mation of MF390 with chromosomal DNA from MF160(thrC::PspoIID–gfp erm).

Plasmids

All plasmid constructions were performed in Escherichia coliDH5a using standard methods.

To construct pMF7 (3¢ part of spoIIGB–gfp spc), the 3¢ endof spoIIGB was amplified by polymerase chain reaction(PCR) with primers oMF4 (5¢-GCCGAATTCGCGGGAGTGAAGCCCT-3¢) and oMF5 (5¢-TGGCTCGAGCACCATTTTGTTGAACT-3¢) that replaced the stop codon with an XhoIsite. The PCR fragment was digested with EcoRI and XhoIand cloned into pKL147 cut with EcoRI and XhoI (Lemon and Grossman, 1998). The gfp coding sequence(GFPmut2) in pKL147 contains the codon substitutions S65T,V68L and S72A (Cormack et al., 1996). To construct pMF6(3¢ part of sigE–gfp kan), the 3¢ end of spoIIGB was clonedbetween the EcoR1 and Xho1 sites of pKL168, a gift from K.Lemon (MIT, USA). To construct pMF19 (PspoIIG–gfp spc), aPCR fragment containing the entire spoIIG promoter (PspoIIG) was amplified from PY79 chromosomal DNA withprimers oMF6 (5¢-CAGCAGGAATTCAGTGATCGTCCGGATGATT-3¢) and oMF7 (5¢-CAGCAGAAGCTTTGCCCACGCTGTTCCCCTT-3¢) and digested with EcoRI andHindIII. A PCR fragment of the gfp coding sequence (with anoptimum ribosome-binding sequence, 5¢-AAGGAGGAA-3¢;Vellanoweth and Rabinowitz, 1992) was amplified frompKL147 DNA (Lemon and Grossman, 1998) with primersoDR107 (5¢-GGCAAGCTTACATAAGGAGGAACTACTATGAGTAAAGGAGAAGAAC-3¢) and oDR78 (5¢-GCCGGATCCTTATTTGTATAGTTCATCCATGCC-3¢), gifts from D. Rudner(Harvard University, USA). The gfp DNA fragment wasdigested with HindIII and BamHI and was cloned into theplasmid pDG1730 (Guerout-Fleury et al., 1996) in a three-way ligation containing the DNA fragment of PspoIIG. To con-struct pMF35 (Pspac

C–gfp spc), the primer pair oMF24(5¢-GCCGAATTCTACACAGCCCAGTC-3¢) and oMF25 (5¢-GCCAAGCTTAACCGGATTCCACACATTATGCCACA-3¢)was used to generate a PCR fragment that included the con-stitutive spac promoter (Pspac

C) from plasmid pAG58 (Jaackset al., 1989). Pspac

C is a modified form of the Pspac promoterthat lacks the operator for the LacI repressor and hence is active constitutively. The Pspac

C promoter fragment wasdigested by EcoRI and HindIII and was cloned into pDG1730(Guerout-Fleury et al., 1996) in a three-way ligation with thegfp DNA fragment (described above). To construct pMF15(PspoIID–gfp erm), spoIID promoter DNA (PspoIID) was preparedfrom pDR102, a gift from D. Rudner (Harvard University), andwas cloned into pDG1664 (Guerout-Fleury et al., 1996) in athree-way ligation with the gfp DNA fragment (describedabove). To construct pMF27 (PspoIIG–lacZ spc), a spoIIG pro-moter fragment (PspoIIG, described above) was cloned intopDG1728 (Guerout-Fleury et al., 1996). To construct pMF46(Pspac

C–spoIIGAB spc), a PCR fragment containing the entirespoIIG operon (both spoIIGA and spoIIGB) was amplifiedfrom PY79 chromosomal DNA with primers oMF23 (5¢-GCCCTCGAGGCGTGAGGCAAGAAAGAAAGG-3¢) and oMF8(5¢-AGTAGTGGATCCTTACACCATTTTGTTGAACTC-3¢).The Pspac

C promoter was amplified using oMF24 (described




above) and oMF35 (5¢-GCCCTCGAGAACCGGATTCCACACATTATGCCACA-3¢) from pAG58 (Jaacks et al., 1989). ThespoIIG operon DNA fragment, digested with XhoI and BamHI,and the Pspac

C DNA fragment, digested by EcoRI and XhoI,were ligated in a three-way ligation into pDG1730 (Guerout-Fleury et al., 1996).

General methods

Bacillus subtilis strains grown in hydrolysed casein growth media at 37∞C were induced to sporulate by theresuspension method (Sterlini and Mandelstam, 1969;Harwood and Cutting, 1990). Time in sporulation is indicatedafter resuspension. Sporulation efficiency was determined as described previously (Rudner et al., 1999). Competentcells of B. subtilis and E. coli were prepared as describedpreviously (Dubnau and Davidoff-Abelson, 1971; Sambrooket al., 1989). Plasmid preparation from E. coli grown inLuria–Bertani medium in the presence of ampicillin(50 mg ml–1) was performed using a QIAprep spin miniprep kit(Qiagen). Assay of b-galactosidase activity was performed asdescribed previously (Rudner et al., 1999).

Fluorescence and time-lapse microscopy

Sporulation was induced according to the method of Sterliniand Mandelstam (1969). At the appropriate time of sporula-tion, 0.5 ml aliquots of the sporulating culture were removed,centrifuged briefly and resuspended in 0.05 ml of 1 ¥ PBSsupplemented with the membrane stain FM 4-64 (MolecularProbes) at 1 mg ml–1 and the DNA stain DAPI (Sigma) at2 mg ml–1, final concentration. This concentrated cell suspen-sion (3 ml) was placed on a microscope slide. A freshly pre-pared poly L-lysine-treated coverslip was used to immobilizethe cells for microscopic analysis.

For time-lapse observation, a chambered slide (VWR Scientific) filled with the sporulation medium containing 1%agarose was used. At the appropriate time after the initiationof sporulation, 0.5 ml aliquots of sporulating culture weretaken, centrifuged briefly and resuspended in 0.05 ml of theoriginal culture supernatant. The concentrated bacteria wereapplied to the agarose bed in the chambered slide, and thena freshly prepared poly L-lysine-treated coverslip was usedto immobilize the cells for microscopic analysis. The temper-ature of the room in which the microscope was located wasset at approximately 25∞C. Minimum exposure times wereused to minimize the illumination of the bacteria. Fluores-cence microscopy was performed as described previously(Eichenberger et al., 2001).

Protoplasting

Protoplasts were prepared as described by Wu et al. (1998).

Immunoblot analysis

Immunoblot analysis was performed according to the proce-dure of Rudner et al. (1999). Monoclonal anti-GFP antibody(Clontech) was used for the detection of pro-sE/sE–GFP.

Affinity-purified anti-pro-sE/sE antibodies were used for thedetection pro-sE/sE.

Acknowledgements

We thank P. Stragier for providing strain MO1780, D. Rudnerfor his gift of PCR primers and plasmid, J. E. Gonzalez-Pastor for his help in making pMF6, and W. Haldenwang, A. E. M. Hofmeister and D. Rudner for many helpful com-ments and discussions. We also thank all members of ourlaboratory. M.F. was supported by a grant-in-aid for researchabroad from the Ministry of Education, Science and Culture,Japan. This work was supported by NIH grant GM18568 toR.L.

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An investigation into the compartmentalization of the sporulation transcription factor σE in Bacillus subtilis