Bacillus subtilis Pro- E Fusion Protein Localizes to the Forespore Septum and Fails To Be Processed When Synthesized in the Forespore

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1 JOURNAL OF BACTERIOLOGY, Aug. 1997, p Vol. 179, No /97/$ Copyright 1997, American Society for Microbiology Bacillus subtilis Pro- E Fusion Protein Localizes to the Forespore Septum and Fails To Be Processed When Synthesized in the Forespore JINGLIANG JU, TINGQIU LUO, AND W. G. HALDENWANG* Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas Received 31 March 1997/Accepted 5 May 1997 Endospore formation in Bacillus subtilis begins with an asymmetric cell division that partitions the bacterium into mother cell and forespore compartments. Mother cell-specific gene expression is initiated by E,a transcription factor that is active only in the mother cell but which existed as an inactive precursor (pro- E ) in the predivisional cell. Activation of pro- E involves the removal of 27 amino acids from its amino terminus. A chimera of pro- E and the green fluorescent protein (GFP) was expressed from either the normal sige promoter (P spoiig ), which places pro- E ::GFP in both mother cell and forespore compartments, or the forespore-specific promoter (P dacf ), which produces pro- E ::GFP only in the forespore compartment. The pro- E ::GFP expressed from P spoiig, but not P dacf, was converted to a lower-molecular-weight form by a mechanism dependent on gene products (SpoIIGA and F ) that are essential for normal pro- E processing. This finding is consistent with the pro- E processing reaction occurring only in the mother cell compartment. In processing-deficient cells, pro- E ::GFP was found to accumulate at the septal membrane, a location where its processing apparatus would be susceptible to triggering from the adjoining forespore. * Corresponding author. Fax: (210) Differentiating cells produce offspring which are endowed with unique developmental fates. The mechanisms by which such cells become programmed to follow a particular path are being unraveled in a number of diverse model systems. One such model is endospore formation by the bacterium Bacillus subtilis, where the simplicity of the sporulation process allows for detailed biochemical and genetic analyses (9, 28). Early in sporulation, B. subtilis partitions itself into two cells of unequal size that are contained within a common outer wall. The smaller of the two cells (forespore) becomes engulfed by the larger mother cell, which then nurtures the forespore during subsequent development. When development is complete, the mother cell lyses, releasing the mature spore into the environment. There is compelling evidence that the distinct fates of mother cell and forespore are orchestrated by compartmentspecific transcription factors that control each cell s individualized program of gene expression (6, 10, 19, 24, 29). Mother cell-specific gene expression is primarily controlled by the sequential appearance of the transcription factors E and K, while forespore-specific genes are activated first by F and then by G (Fig. 1) (reviewed in references 1, 2, 3, and 28). Both F and E are synthesized at the onset of sporulation (prior to the appearance of the sporulation septum) but are held inactive until after the septation event (1, 5, 12, 18, 20, 25, 27, 31). The mechanisms for silencing sigma factor activity until compartmentalization occurs are unique for each sigma factor. F is sequestered into an inactive complex by an anti- F protein (SpoIIAB) (1, 5, 25, 27), while E is synthesized as an inactive proprotein which is activated when a developmentally regulated protease removes 27 amino acids from its amino terminus (12, 20). Each of the two inactive sigma factors, distributed throughout the cell prior to septation, is presumably present in both compartments after septation. The asymmetric cell division triggers the release of F from its inhibitor in the forespore (2, 7, 8) and the appearance of E -dependent gene expression in the mother cell (6, 12). This report addresses the hypothesis that E activity is restricted to the mother cell because the pro- E processing reaction is confined to that compartment. MATERIALS AND METHODS Plasmids and bacterial strain constructions. The B. subtilis strains and plasmids used in this study are listed in Table 1. psel-1 is a plasmid carrying a 340-bp PstI-Sau3A fragment that encodes 165 bp of the 5 end of sige joined to PstI-BamHI-cut psgmu31 2 (15). A 420-bp HindIII fragment from ppp212 (32) containing the dacf promoter was cloned into the HindIII site of psel-1 to create pfel-1. The proper orientation of the dacf promoter relative to sige was verified by restriction endonuclease digestions. The gfp gene, released from plasmid pcw33 (30) as a 768-bp SmaI-ClaI fragment, was blunt ended with the Klenow fragment of DNA polymerase I and ligated into large SalI fragment of pfel-1 that had been made blunt with the Klenow fragment of DNA polymerase I. The resulting plasmid, pf1, contains a sige55::gfp in-frame fusion under the control of the dacf promoter. pf1 was integrated into the chromosome of SMY cells through a single-site (Campbell-like) recombination. Integrants were selected on the basis of the plasmid-encoded chloramphenicol resistance (5 g/ml). Integration of the plasmid could occur at spoiig or dacf. Transformants in which the integration had occurred at one or the other site were distinguishable by their phenotypes on Difco sporulation (DS) medium. Integration at spoiig (SPF1) confers a Spo phenotype due to the disruption of spoiig, while cells with an integration at dacf (SPF2) are Spo. The F structural gene in SPF1 was disrupted by transformation with chromosomal DNA from EUR9030 (spoiiac::erm) and selection for erythromycin resistance (Erm r ). This procedure yielded strain SPF3. Strain SPF4 was constructed by transforming PEW with EUR9030 chromosomal DNA and selection for resistance to erythromycin (1 g/ml). This procedure yielded PEWF (spoiiga 5 spoiiac::erm). Plasmid pf1 (P dacf ::sige55::gfp cat) was integrated into the chromosome of PEWF cells by transformation and selection for chloramphenicol (5 g/ml) resistance (Cm r ). PEWF is SigF. Thus, transformants in which the P dacf ::sige55::gfp integrated at dacf would not be able to express green fluorescent protein (GFP), while those in which the fusion integrated at spoiig would be able to synthesize GFP. Cm Erm clones were incubated in DS medium at 30 C for 24 h and examined microscopically for GFP fluorescence. PEWF-1 was selected as a GFP clones (i.e., pf1 integrated at spoiig). The chromosomal DNA was isolated from PEWF-1 and transformed into SMY to separate the spoiiga 5 P spoiig ::sige55::gfp construction from spoiiac::erm. Cm r clones were screened for Erm s (SpoIIAC ) colonies. This procedure yielded SPF4 (P spoiig ::sige55::gfp spoiiga 5). 4888

2 VOL. 179, 1997 LOCALIZATION OF B. SUBTILIS PRO- E FUSION PROTEIN 4889 FIG. 1. Sequential activation of transcription factors during sporulation. At the onset of sporulation (t 0 ), the first two sporulation-specific factors are synthesized as either an inactive precursor (pro- E )ora factor ( F ) that is inactivated by association with an anti- protein (SpoIIAB). By the second hour into sporulation (t 2 ), an asymmetric cell division establishes two distinct cell compartments, and each of the previously inactive factors becomes active in only one of the two compartments: E in the mother cell and F in the forespore. These transcription factors establish the initial patterns of compartment-specific gene expression. By 2 h later (t 4 ), the forespore has been engulfed within the mother cell and two additional compartment-specific sigma factors ( K and G ), dependent on the earlier sigma factors for their synthesis, appear and augment or replace E and F to direct late spore gene expression. Sporulation is completed with the death of the mother cell and release of the mature spore approximately 8 h after sporulation onset (t 8 ). Visualization of fluorescence. Fluorescence was visualized as described previously (3, 21, 30). Culture samples of 200 l were transferred to 1.5-ml microcentrifuge tubes on ice; 50 l of preservation buffer (40 mm NaN 3, 50% sucrose, 0.5 M Tris [ph 7.5], 0.77 M NaCl) was added, and the suspension incubated at 4 C for at least 2 h. A 3- l aliquot of the cell suspension was mixed with 1 l of 4,6-diamidino-2-phenylindole (DAPI; 1 g/ml; Sigma) on a slide precoated with 0.01% polylysine (Sigma) and covered with a polylysine-coated coverslip. Cells were viewed with a Zeiss Axiophot epifluorescence microscope with a 100-W mercury lamp source and a 100 Plan-Neofluar oil immersion objective lens. Images were recorded on Ektachrome 1600 slide film. The images were scanned and prepared with Adobe Photoshop version 4.0. GFP fluorescence was viewed with a chroma fluorescein isothiocyanate filter set (Chroma Technology) ( exciter filter, FT510 chromatic beam splitter, and LP520 barrier filter). For pictures taken with simultaneous phase-contrast and GFP fluorescent light, the phase-contrast light level was adjusted so that the light was bright enough to show the cells but dim enough that the GFP fluorescent light could still be seen. Finally, DAPI-stained images were obtained with a chroma fluorescein isothiocyanate filter set (Chroma Technology) (BP 365/12 exciter filter, FT395 chromatic beam splitter, and LP397 barrier filter). Images of the same field were obtained under conditions for recording GFP, GFP-plusphase-contrast, and DAPI fluorescence. The camera was set at the auto mode for all pictures. Western blot analysis. Crude cell extracts were prepared, as described previously (20), by disrupting bacteria with a French pressure cell. The protein concentration was determined with a Bio-Rad protein assay as instructed by the manufacturer. Extracts were fractionated by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose and blocking of the nitrocellulose with BLOTTO, the protein bands were probed with an anti-gfp monoclonal antibody (Clontech Laboratories, Inc., Palo Alto, Calif.). Bound antibody was visualized with an alkaline phosphataseconjugated goat immunoglobulin against mouse immunoglobulin (American Qualex), using CDP-Star (Boehringer Mannheim) as the substrate reagent. Induction of sporulation. Cells were grown in DS medium at either 30 or 37 C. The level of GFP fluorescence signal is higher at 30 C (3); 30 C was therefore used for the fluorescence studies. General methods. DNA manipulations and the transformation of Escherichia coli were done according to standard protocols. Transformation of competent B. subtilis cells was carried out by the method of Yasbin et al. (33), with transformants selected on LB agar plates contain 1 g of erythromycin or 5 g of chloramphenicol. Sporulation agar plates consisted of DS medium solidified with 1.5% Difco Bacto Agar. RESULTS AND DISCUSSION Western blot analysis of pro- E ::GFP accumulation and processing. GFP of the Pacific jellyfish Aeguorea victoria is a powerful fusion tag for protein localization studies and has been used in B. subtilis to visualize compartment-specific gene expression (3, 21, 30). As described in Materials and Methods, we joined the coding sequence for the first 55 amino acids from the amino terminus of the pro- E structural gene (sige), in frame, to the coding sequence for GFP to create a chimeric sige55::gfp gene. The pro- E element in this fusion contains the pro- E cleavage site and 28 amino acids from the amino terminus of mature E. These 55 amino acids, when joined to heterologous proteins, allow such proteins to be recognized and processed by the B. subtilis developmentally regulated protease (4). Processing of pro- E55 ::GFP can be monitored by Western blot analyses, while localization of the fusion protein can be observed by fluorescence microscopy. sige55::gfp was first placed into B. subtilis under the control of the promoter (P spoiig ) which normally directs transcription of sige. Crude extracts were prepared from sporulating cultures of this strain (SFP1) and examined for pro- E55 ::GFP accumulation and processing by Western blot techniques. As expected, full-length pro- E55 ::GFP is seen first, followed by the appearance of a faster-migrating form as the culture proceeds into sporulation (Fig. 2A). The apparent size of the smaller pro- E55 ::GFP and the time in development when it appears strongly suggest that this protein is the processed form of the pro- E55 ::GFP chimera. To confirm this notion, the P spoiig ::sige55::gfp fusion was introduced into B. subtilis strains with mutations in genes (spoiiga and spoiiac) that are essential for normal pro- E processing (14, 16). In these genetic backgrounds, the processed form of pro- E55 ::GFP was no longer detectable in Western blot analyses (Fig. 2C and D). We conclude that the pro- E55 ::GFP, when expressed from the spoiig (i.e., sige) promoter, is recognized and cleaved by the pro- E processing apparatus. We next placed the sige55::gfp fusion under the control of the forespore-specific dacf promoter and examined its expression in sporulating B. subtilis. Western blot analyses revealed that the chimeric GFP precursor accumulated but failed to be processed into the lower-molecular-weight form when expressed from this promoter (Fig. 2B). Apparently, pro- E55 ::GFP, and by analogy pro- E itself, cannot be processed when synthesized within the forespore. This observation is consistent with the notion that restriction of E activity to the mother cell is due to selective pro- E processing in only that compartment. Strain or plasmid TABLE 1. Bacterial strains and plasmids used Relevant genotype or features Source, construction, or reference B. subtilis strains SMY trpc Laboratory strain PEW spoiiga 5 Laboratory strain EUR9030 spoiiac::erm 4 PEWF spoiiga 5 spoiiac::erm EUR90303PEW PEWF-1 spoiiga 5, spoiiac::erm pf13pewf P spoiig ::sige55::gfp SPF1 P spoiig ::sige55::gfp pf13smy (Spo ) SPF2 P dacf ::sige55::gfp pf13smy (Spo ) SPF3 P spoiig ::sige55::gfp spoiiac::erm EUR90303SPF2 (Spo ) SPF4 P spoiig ::sige55::gfp spoiiga 5 pewf13smy (Spo ) Plasmids ppp212 bla dacf 32 psgmu31 2 bla cat lacz 15 pcw33 bla km gfp 30 psel-1 bla cat sige::lacz 15 pfel-1 bla cat P dacf ::sige::lacz This study pf1 bla cat P dacf ::sige::glp This study

3 4890 JU ET AL. J. BACTERIOL. FIG. 2. Western blot analysis of pro- E55 ::GFP accumulation in sporulating cells. Cells were grown in DS medium and harvested at the end of exponential growth, i.e., the onset of sporulation (lane 1), or 1.5-h intervals thereafter (lanes 2 to 5). Total protein from sporulating cells was fractionated on 12% polyacrylamide gels containing sodium dodecyl sulfate, transferred to nitrocellulose membranes, and probed with an anti-gfp monoclonal antibody (Clontech). Bound antibody was detected with X-ray film, using a secondary antibody conjugated to alkaline phosphatase (American Qualex) and a chemoluminescent substrate (CDP-Star; Boehringer Mannheim). (A) P spoiig ::sige55::gfp in a processingproficient strain (SFP1); (B) P dacf ::sige55::gfp in a processing-proficient strain (SFP2); (C and D) P spoiig ::sige55::gfp in processing-deficient SpoIIAC (SFP3) and SpoIIGA (SFP4) strains, respectively. Lane 6 in panel A represents a congenic wild-type strain (SMY) without the sige55::gfp fusion; lanes 6 in panels B to D represent P spoiig ::sige55::gfp in a processing-proficient strain at 4.5 h after the end of exponential growth (t 4.5 ). The arrowheads indicate the positions of the unprocessed (pro-gfp) and processed (GFP) fusion proteins. Localization of pro- E55 ::GFP in sporulating B. subtilis. The B. subtilis strains that expressed sige55::gfp from either P spoiig or P dacf were next examined by fluorescence and phase-contrast microscopy to verify the location of the pro- E55 ::GFP synthesis. It should be noted that the recombination event, by which sige55::gfp integrated into the B. subtilis chromosome behind P spoiig, placed the sige55::gfp plasmid between the wild-type sige allele and P spoiig. Thus, with sige separated from its promoter, none of these strains could synthesize a functional pro- E. As a consequence of this, they are sporulation deficient and do not progress beyond stage II (i.e., the forespore is not engulfed by the mother cell). Instead, a second septum is laid down at the pole of the cell opposite that at which the first septum appears (13). This disporic morphology, with two forespore compartments bracketing the former mother cell, is characteristic of SigE cells and is evident in the micrographs of the P spoiig ::sige55::gfp strains (Fig. 3A, C, and D). Figures 3A 1 to A 3 and A 4 to A 6 are series of micrographs in which two representative groupings of cells were photographed under different illumination conditions. These micrographs illustrate P spoiig ::sige55::gfp-expressing cells, which could process the pro- E55 ::GFP product. Figures 3A 4 to A 6 are images at a higher magnification to emphasize detail. The images in Fig. 3A 1 and A 4 depict both phase-contrast illumination and GFP fluorescence. The phase-contrast aspect is eliminated in Fig. 3A 2 and A 5 to maximize visualization of GFP fluorescence. The images in Fig. 3A 3 and A 6 illustrate DAPI staining of these same cells to detect chromosomal DNA. DAPI staining of these cells occurs predominantly at the two polar forespore compartments. The positions of the forespore compartments of the same cell in each group are indicated by arrowheads in Fig. 3A 1 to A 3 and arrows in Fig. 3A 4 to A 6. The GFP provided diffuse fluorescence that was most evident within the central compartment between the polar septa. This is best seen in Fig. 3A 4 to A 6. In the B. subtilis strain carrying the P dacf ::sige55::gfp fusion, all of the sporulation essential genes are intact. Therefore, unlike the strain discussed above, it is able to proceed normally through the sporulation process and develop a single forespore compartment which is engulfed by the mother cell. Also, GFP fluorescence in the P dacf ::sige55::gfp strain is essentially limited to the forespore compartment (Fig. 3B). The arrows in this series of figures indicate the positions of the mother cell and forespore compartments of a single cell in each series. Figure 3B 1 to B 6 illustrate the GFP fluorescence of representative cells with and without phase-contrast illumination to display the cell outline. DAPI staining of these same cells stained both nucleoids but preferentially highlighted that of the mother cell compartment. The reduced staining of the forespore compartment was variable and presumably represents impaired penetration of the dye into the less permeable forespores. The GFP fluorescence data in Fig. 3A and B verify that the pro- E55 ::GFP, synthesized from the spoiig promoter and processed into a lower-molecular-weight form, accumulated predominantly in the mother cell compartment, while that synthesized by transcription from P dacf and not processed was localized within the forespore compartment. We next looked at pro- E55 ::GFP localization in strains that carried the P spoiig ::sige55::gfp fusion and an additional mutation (i.e., spoiiga 5 or spoiiac::erm) that blocks the pro- E processing reaction. These strains, like SFP1 in Fig. 3A, are arrested at stage II of sporulation and have a disporic terminal phenotype with two polar septa. Figures 3C 1 to C 9 display representative cells from a P spoiig ::sige55::gfp spoiiac::erm culture, visualized as in the previously discussed series of micrographs. spoiiga 5 cells carrying a similar gfp fusion are illustrated in Fig. 3D 1 to D 9. As was the case for the processing-competent P spoiig ::sige55::gfp-containing strain (Fig. 3A), the GFP fluorescence in the processing-deficient strains is observed predominantly in the central compartment; however, unlike the former strain, the processing-deficient strains had a further enrichment of the GFP at the positions of the septal membranes. These regions are indicated, in representative cells, by arrows in Fig. 3C 1 to C 6 and D 1 to D 6. We interpret the presence of GFP at the septa of processing-deficient B. subtilis, but not the processing-competent strain, as evidence that the septal localization of the GFP is a consequence of its fusion to the SigE pro sequence and that it represents an association that exists prior to processing. Presumably, the pro- E55 ::GFP also binds to the septum of the processingcompetent B. subtilis; however, in this strain the pro sequence is cleaved, releasing the processed GFP into the cell interior. We speculate that in the absence of processing, the pro- E55 ::GFP accumulates at the septal membranes. The fluorescence micrographs of the processing-deficient strains show a diverse pattern of GFP localization. In some cases (Fig. 3C 4 to C 6 and D 4 to D 6 ) most of the fluorescence is associated with the septa, while other cells (Fig. 3C 7 to C 9 and D 7 to D 9 ) display more intense fluorescence throughout the central cell compartment. Our Western blot data (Fig. 2C and D) show no evidence of pro- E55 ::GFP processing in these strains; however, they do show smaller protein bands which reacted with the antibody. These likely represent GFP degradation products. It is possible that some of these breakdown products lost the pro- E element but still retained the capacity for fluorescence. We were unable to test this in our Western blot experiment. In our hands, and as stated in the manufacturer s (Clontech s) product analysis certificate, GFP does not fluoresce under this condition. If the lower-molecular-weight GFP fragments could fluoresce, Pro- E55 ::GFP turnover would allow individual cells to exhibit distinct patterns of whole-cell fluorescence, depending on the degree of pro- E55 ::GFP proteolysis in each cell. Alternatively, the variable pattern may be a reflection of a limited number of pro- E

4 VOL. 179, 1997 LOCALIZATION OF B. SUBTILIS PRO- E FUSION PROTEIN 4891 FIG. 3. Localization of Pro- E55 ::GFP in sporulating B. subtilis. Stationary-phase B. subtilis cells were diluted 1/200 into DS medium and incubated for 12 h at 30 C. Samples were treated as described in Materials and Methods to maximize GFP fluorescence, stained with DAPI, and photographed under phase-contrast-plusfluorescence microscopy to visualize the cells plus GFP fluorescence (A 1 and A 4,B 1 and B 4,C 1,C 4, and C 7 ;D 1,D 4, and D 7 ), fluorescent conditions to highlight GFP (A 2 and A 5 ;B 2 and B 5 ;C 2,C 5, and C 8 ;D 2,D 5, and D 8 ), and fluorescent conditions to detect DAPI staining (A 3 and A 6 ;B 3 and B 6 ;C 3,C 6, and C 9 ;D 3,D 6, and D 9 ). (A) SFP1 (P spoiig ::sige55::gfp, processing proficient); (B) SFP2 (P dacf ::sige55::gfp, processing proficient); (C) SFP3 (P spoiig ::sige55::gfp spoiiac::erm, processing deficient); (D) SFP4 (P spoiig ::E55::gfp spoiiga 5 processing deficient). The arrows indicate the positions of the forespore compartments (A), forespore and mother cell (B), and forespore septa (C and D). target sites in the forespore septum and differences in the amount of pro- E55 ::GFP synthesized by individual cells at the time the micrograph was taken. In the absence of processing, pro- E ::GFP could accumulate and saturate a putative limited number of membrane attachment sites. Any additional pro- E55 ::GFP would then remain free within the mother cell. The cells used in the microscopic study were grown in DS medium at 30 C to maximize the fluorescence of GFP. We could not detect fluorescence of cells carrying the P spoiig ::sige55::gfp fusion before 10 h after resuspension in DS medium (t 10 ). By this time, the cells within the culture had left exponential growth and had begun sporulating approximately 1 h earlier. If the strain carried the P dacf ::sige55::gfp fusion, fluorescence was not evident before 12 h. Representative quantitative data for the fluorescence patterns of the cells in these cultures are presented in Table 2. At the earliest time (t 10 ) when we detected GFP in the P spoiig ::sige55::gfp strains, enrichment of GFP at the septum was rare ( 4% of the fluorescing cells) in the processing-competent strain but common ( 80% of the fluorescing cells) in the processing-deficient strains (Table 2). This may reflect rapid processing and release of the pro- E55 ::GFP from the septum. Although such a model is plausible, it is possible that the pro- E55 ::GFP was released from the septum of the processing-competent strain after the cells were harvested and were incubated at 4 C to allow the GFP to assume its fluorescent structure. Regardless of which possibility is correct, there is a clear difference in the likelihood of whether GFP will be found at the septa, depending on whether the SigE pro sequence can be cleaved from it. We did not see localization of pro- E55 ::GFP at the septa of cells expressing sige55::gfp fusion from P dacf. At the earliest times that we could visualize the GFP fluorescence in such a strain (t 12 ), the signal was dispersed within the forespore. Although this finding raises the intriguing possibility that pro- E ::GFP does not bind the septum when synthesized in the forespore compartment, we cannot say whether our failure to detect such a localization is due to the absence of pro- E55 ::GFP binding or a masking of the binding by relatively high levels of dispersed GFP in this small compartment. The pro element encodes an amino acid sequence capable of forming an amphipathic helix with a highly charged face (26). As such, it is unlikely to be able to directly insert itself into a biological membrane. Instead, is more likely to associate with a protein already in the membrane. The tethering of the pro- E55 ::GFP to the septal membrane does not depend on any protein now known to be needed for the processing reaction (i.e., SpoIIGA or a F -dependent gene product). The hypothetical pro- E binding protein may not even be sporula-

5 4892 JU ET AL. J. BACTERIOL. TABLE 2. GFP fluorescence patterns Strain Fluorescence a indicated culture age b No. of cells counted at 10 h 12 h 14 h 16 h 18 h SFP1 (P spoiig ::gfp) N S W P SFP2 (P dacf ::gfp) N S W P SFP3 (P spoiig ::gfp spoiiac::erm) SFP4 (P spoiig ::gfp spoiiga 5) N S W P N S W P a N, no or very weak fluorescence; S, fluorescence enhanced at septa; W, whole-cell fluorescence (10-h cultures)/central compartment fluorescence (12-h and later cultures); P, prespore fluorescence. b Cultures, grown to stationary phase in LB, were diluted 1/300 into DS medium and incubated at 30 C. Sporulation onset was approximately 9 h postdilution into DS medium. tion specific but may instead be a vegetative cell protein normally associated with cell division and septation in general. In support of such a notion, pro- E has been found to copurify with cell membranes when expressed in vegetatively growing B. subtilis (11). Both pro- E and its putative processing enzyme (SpoIIGA) are synthesized at the onset of sporulation; however, E activation does not occur until approximately 1 h later when the septation event establishes the two cell compartments (9, 28). The triggering molecule for pro- E processing has recently been discovered to be an extracellular signaling protein (SpoIIR) (12, 17, 22) that is synthesized within the forespore compartment (34). Hofmeister et al. proposed that SpoIIGA, the pro- E processing enzyme, may be a two-domain protein that resides within the forespore septum, with one domain serving as the pro- E protease and the second serving as a signal transduction domain that responds to transmembrane FIG. 4. Model of pro- E activation. SpoIIGA and pro- E are synthesized prior to septation and should be present in both compartments. SpoIIR is synthesized predominantly if not exclusively in the forespore (34). As proposed by several investigators (12, 17, 22), SpoIIR, synthesized under the control of F containing RNA polymerase (E F ) in the forespore, activates the putative pro- E processing enzyme, SpoIIGA (GA). Pro- E, presumably tethered by its pro sequence to an undefined entity within the septal membrane (X), encounters activated SpoIIGA, which cleaves its pro sequence and releases E into the mother cell. It is unclear whether limiting the processing of pro- E only in the mother cell could be due to the polarity of a transmembrane SpoIIR-SpoIIGA interaction or, alternatively, a difference in the state of SpoIIGA or X in each compartment. activation by SpoIIR (12). This signal would trigger the protease domain to cleave pro- E into mature E. They further hypothesized that this process could occur vectorally, thereby directing the processing reaction to only the mother cell compartment. Our data support such a model (Fig. 4). The observed accumulation of pro- E ::GFP at the septal membrane places pro- E at a site where it could be readily processed in response to a putative transseptal signal. Although this model is intriguing, the notion that SpoIIR must act vectorally to activate pro- E processing is not without controversy. Zhang et al. (34) observed that E activation was limited to the mother cell compartment even when SpoIIR was synthesized from an alternative promoter prior to septation and therefore presumed to be present in both compartments. These authors suggest that a E activation factor other than SpoIIR, possibly SpoIIGA in a particular orientation, could be restricted to the mother cell. Regardless of the mechanism involved, our finding that pro- E processing appears to occur only in the mother cell supports the general model that pro- E processing is compartment specific and explains why E -dependent transcription is observed only in the mother cell. 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