INTRODUCTION

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Nutrient deprivation of the gram-positive bacterium Bacillus subtilis triggers asymmetric cell division, a landmark event of the sporulation process. The smaller progeny cell, the forespore, becomes engulfed by the larger one, the mother cell, which transiently functions as a nurse cell before lysing to release the mature spore (26, 30). Although synthesized prior to septation and partitioned into both the forespore and the mother cell, the transcription factor ^F becomes active only in the forespore, therein initiating a genetic program that culminates in the formation of the dormant spore and launching by intercellular signaling the developmental program of the mother cell (17).

The specific release of ^F activity in the forespore is controlled by the complex interaction of three regulatory proteins, SpoIIAA, SpoIIAB, and SpoIIE, in conjunction with the formation of the sporulation septum (1, 9, 19, 22). Although the subject of intense investigation, the precise mechanisms that restrict ^F activity to the forespore are not yet fully understood (10, 15). In order to gain further insight into that important regulatory step, we have isolated mutations enhancing ^F activity. One previously identified class of ^F-activating mutations is characterized by the formation of abortive forespore compartments at both poles of the sporulating cell, the so-called disporic phenotype that is easily recognizable by phase contrast microscopy (26). The twofold increase of ^F activity, as monitored by ^F-dependent lacZ fusions carried by these mutants, is due to the activation of ^F in both forespore compartments (18). By screening for mutations enhancing ^F activity and accompanied by a nondisporic Spo phenotype, we have identified a new locus in which some mutations block sporulation shortly after completion of the polar septum. Subsequent characterization of this locus indicated that it encodes two proteins, one of which prevents the lethal action of the other, with no direct bearing on the ^F activation cascade.

	MATERIALS AND METHODS

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Bacterial strains and techniques. All B. subtilis strains were derivatives of the sporulation-proficient strain JH642 (trpC2 pheA1), with the exception of the xin15 L8460 strain obtained from D. Karamata. The reporter lacZ fusions to the spoIIR, spoIIQ, spoIID, and spoIIIG promoters have been described (20, 21, 29, 31), as have the gfp fusions to the spoIIQ and cotE promoters (20, 33). For monitoring sporulation efficiency, cells were grown at 37°C for about 40 h in Difco sporulation (DS) medium (28) and the number of spores was determined by their resistance to heat killing (10 min at 80°C). -Galactosidase was assayed as previously described (29) and is expressed as nanomoles of 2-nitrophenyl--D-galactopyranoside hydrolyzed per minute per milligram of protein. Synthesis of green fluorescent protein from Aequorea victoria was monitored by fluorescence microscopy according to published protocols (33). UV mutagenesis of a wild-type strain containing a spoIIR-lacZ fusion at the amyE locus was performed according to standard protocols (8), and cells were directly plated on DS agar plates containing 400 µg of 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)/ml. Spontaneous Spo⁺ suppressor mutations obtained in a spoIISB null strain carrying an extra copy of spoIISA at amyE were identified on DS agar plates by the characteristic brown pigmentation of sporulating B. subtilis colonies. These mutations were mapped to spoIISA by their linkage with the tetracycline resistance marker in spoIISB or the chloramphenicol marker at amyE as assessed by DNA transformation. Antibiotic-resistance cassettes and conditions of selection for antibiotic resistance have been described (12).

Cloning the spoIIS locus. Sporulation-deficient mutants exhibiting enhanced ^F activity were transformed with an integrative plasmid library (23), selecting for chloramphenicol resistance, and screened for sporulation recovery as judged by their pigmentation phenotype on DS agar plates. Prior to that step the spoIIR-lacZ fusion, associated in the mutants with a chloramphenicol resistance marker at amyE, was removed and replaced with a spectinomycin resistance marker (11). Rescuing plasmids could be recovered in Escherichia coli, and their insert could be characterized as previously described (21). Two plasmids were isolated that were able to correct the defect of the spoIIS mutants. In one plasmid the Sau3A insert did not extend further upstream than codon 7 in spoIISA, whereas the insert of the other plasmid contained 370 bp upstream of the spoIISA reading frame. The smaller insert contained 261 bp downstream of spoIISB and was followed by a Sau3A insert from another region of the B. subtilis chromosome, with a HindIII site located 41 bp after the junction between the two Sau3A fragments. Various subfragments from these two plasmids were cloned in integrative vectors that could recombine by single crossover at the spoIIS locus (24) or in vectors allowing ectopic integration by a double recombination event at amyE or thrC, occasionally after fusion to lacZ (11). Point mutations in spoIIS were mapped by using a series of overlapping integrative plasmids. They were cloned either by recovery in E. coli of a spontaneously excised plasmid (unable to rescue the original mutation) or by a chromosomal walk from a plasmid integrated in the vicinity of the mutation.

Manipulating the spoIIS locus. A null mutation in spoIISA was created by replacing the 352-bp NdeI-HpaI fragment internal to spoIISA with a kanamycin resistance cassette. A null mutation in spoIISB was created by inserting a tetracycline resistance cassette in the DraI site located at codon 17 in spoIISB. A complete deletion of the spoIIS locus was constructed by cloning a kanamycin resistance cassette between PCR-amplified fragments bracketing an interval extending 48 bp upstream of spoIISA and 76 bp downstream of spoIISB. Because an intact spoIISA gene could not be cloned in E. coli without spoIISB, the spoIISA gene was introduced in two steps at the ectopic amyE site. First, a truncated spoIISA gene starting at codon 7 of spoIISA and extending to codon 17 of spoIISB was introduced at amyE with selection for spectinomycin resistance. Then, a complete spoIISA gene was reconstituted by recombination with a fragment containing 370 bp upstream of the spoIISA reading frame as well as 91 codons of spoIISA, exchanging the spectinomycin marker for a chloramphenicol resistance marker. A similar two-step strategy was followed to put the spoIISA gene under the control of foreign promoters by previously fusing a subfragment containing only 52 bp upstream of the spoIISA reading frame and 91 codons of spoIISA to the desired promoter-bearing fragment. The spoIID promoter (a 290-bp HindIII-PvuII fragment) and the spoIIQ promoter (a 566-bp SphI-Sau3A fragment) have been previously described (20, 29). A 1.5-kb fragment containing the xylA promoter and the xylR gene was a kind gift from F. Arigoni. The spoIISB gene was introduced at the ectopic amyE and thrC sites, either under the control of its own promoter as a 948-bp NaeI-HindIII fragment or under the control of foreign promoters as a 696-bp NdeI-HindIII fragment.

Ultrastructural studies. Samples for electron microscopy were processed as described previously (3). Stained thin sections were examined and photographed on a JEOL-100 CX electron microscope. For quantification of morphological classes, at least 100 complete longitudinal sections were scored from random fields for each sample.

	RESULTS

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Identification of the spoIIS locus. A B. subtilis strain carrying a spoIIR-lacZ transcriptional fusion was UV mutagenized to 98% killing, and cells were plated directly on DS agar plates containing the chromogenic -galactosidase substrate X-Gal and incubated at 37°C. The spoIIR gene is expressed from a weak ^F-controlled promoter (14, 21), and colonies harboring a spoIIR-lacZ fusion become barely blue on such plates. The presence of a mutation leading to the disporic phenotype markedly increases the blue color after 2 days at 37°C, a feature that made that fusion ideal for our genetic screen. Forty-five colonies with a darker blue color were isolated from 150,000 colonies recovered after mutagenesis. Twelve of them were defective in sporulation, with sporulation efficiencies ranging from 10² to 10⁷ of that observed with a wild-type strain. None of these mutants appeared to produce disporic cells as judged by phase contrast microscopy.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Characterization of the spoIIS locus. The genetic organization of the spoIIS region is shown at the top, with partial or complete open reading frames displayed as thick arrows. Asterisks indicate the locations of the mut9 and mut14 mutations. In the simplified physical map, the bordering restriction sites originate from the plasmids used for cloning the region, either from the vector backbone (EcoRI) or from an additional genomic fragment present in the insert (HindIII). The two fragments fused to lacZ for monitoring promoter activity are shown with the presumed positions of the transcription starts (thin arrows). Thick bars in the bottom part of the figure indicate the extents of the DNA fragments that were used for complementation analysis of the two spoIIS mutants, with the results shown in the right-side columns. When these fragments were cloned in integrative plasmids, correction of the sporulation defect (indicated as +) was observed in a variable proportion of the recombinants, depending on the location of the mutation relative to the fragments. Conversely, introducing these DNA fragments at the ectopic amyE locus led to a homogeneous population of transformants. Partial restoration of sporulation of the mut9 strain is indicated by (+).

View larger version (86K): [in this window] [in a new window]   FIG. 2.   The two SpoIIS proteins. A schematic representation of the SpoIISA and SpoIISB proteins (248 and 56 residues, respectively) is shown with the coordinates of the three putative transmembrane domains of SpoIISA. The topological model for SpoIISA is based on the predictions of the TopPred II program (6) and includes the presence of six positively charged residues between the first two transmembrane segments.

Epistatic relationship between SpoIISA and SpoIISB. The SpoIISB translation start codon overlaps the spoIISA translation stop codon, a strong indication that the two genes constitute an operon. It was therefore unexpected that the mut14 mutation in spoIISB can be complemented in trans by a DNA fragment containing an intact spoIISB cistron but extending only up to the NaeI site located at codon 91 of spoIISA (Fig. 1). A shorter DNA fragment extending only up to the NdeI site located at codon 175 of spoIISA does not complement the mut14 mutation (Fig. 1), suggesting that a promoter allowing expression of spoIISB is present in the NaeI-NdeI interval. Indeed, this region of the chromosome is able to drive -galactosidase synthesis when cloned upstream of a promoterless lacZ gene (Fig. 3). The spoIISB promoter is probably recognized by the major vegetative sigma factor ^A as suggested by the variations of its activity during exponential growth and sporulation (Fig. 3). These variations are not significantly altered by the absence of transcription factors involved in the transition to stationary phase, such as ^H, Spo0A, or AbrB (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Expression of spoIIS-lacZ. The specific activity of -galactosidase was monitored in a wild-type strain containing a transcriptional spoIIS-lacZ fusion, either from the PA promoter () or from the PB promoter (), as defined in Fig. 1. Both fusions were inserted at the amyE locus. Bacteria were induced to sporulate by exhaustion in DS medium at 37°C, with the onset of sporulation defined as the time when cultures deviate from exponential growth.

Sporulation phenotype of spoIIS mutants. The strains carrying the spoIISA(mut9) mutation, the spoIISB(mut14) mutation, or the spoIISB null allele behave similarly when grown in sporulation medium (Fig. 4). They do not exhibit any obvious defect during exponential growth, and they reach stationary phase with the same optical density. However, about 2 h after the onset of sporulation the spoIIS mutants show a sudden drop in optical density, down to about 55% of the density of the wild type, a very unusual feature for a sporulation mutant.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Death of spoIIS mutants in stationary phase. The optical density of cells grown in DS medium at 37°C was monitored during exponential growth and sporulation. Strains contained either a wild-type spoIIS locus (), the spoIISA(mut9) mutation (), or the spoIISB null mutation (). The strain containing the spoIISB(mut14) mutation behaved exactly as the spoIISA(mut9) mutant and, for clarity, its results are not shown.

View larger version (154K): [in this window] [in a new window]   FIG. 5.   Morphological consequences of SpoIISA activity. (A) spoIISB cells were grown in DS medium and harvested 2 h (top) or 4 h (bottom) after the onset of sporulation. (B) Cells of a wild-type strain containing an extra copy of spoIISA under the control of the forespore-specific spoIIQ promoter were grown in DS medium and harvested 2 h (top) or 4 h (bottom) after the onset of sporulation. (C) Cells of a wild-type strain containing an extra copy of spoIISA under the control of the xylA promoter were grown in LB medium without xylose (left) or in the presence of 5 mM xylose (added at an optical density at 600 nm of 0.5) and harvested 1.5 h after xylose addition (right). Representative examples of cellular morphologies are shown. Arrowheads point to plasmolysis zones where the cytoplasmic membrane appears to be detached from the cell wall. Thin arrows indicate holes in the peptidoglycan layer. Bars represent 0.3 µm.

Site of action of SpoIISA. Since the absence of SpoIISB does not become apparent until cells enter sporulation, we wondered whether a spoIISB mutation would be corrected in cells synthesizing SpoIISB only during sporulation. Moreover, since spoIISB cells reach stage II after synthesis of the sporulation septum and activation of ^F and ^E, the spoIISB mutation could be rescued either in the forespore or in the mother cell by placing spoIISB under the control of a ^F- or a ^E-dependent promoter. The sporulation defect of the spoIISB null strain is fully corrected when SpoIISB is synthesized in the mother cell under the control of the spoIID promoter, whereas synthesis of SpoIISB in the forespore under the control of the spoIIQ promoter has no effect (Table 2). This result indicates that SpoIISA is acting mainly, if not exclusively, in the mother cell compartment of the sporulating cell. Interestingly, additional synthesis of SpoIISA in the mother cell from the spoIID promoter in a wild-type strain has no effect on sporulation (Table 2), suggesting the presence of an excess of SpoIISB molecules in the cell. However, it should be noted that the P_spoIID-spoIISA hybrid gene has only a 20-fold negative effect on the sporulation of a strain lacking the whole spoIIS locus (Table 2) and might therefore not be fully functional.

View larger version (16K): [in this window] [in a new window]   FIG. 6.   SpoIISA-induced death during exponential growth. Cells containing an extra copy of spoIISA at the amyE locus under the control of the xylA promoter were grown in LB medium at 37°C, and their optical density was monitored before and after addition of 5 mM xylose (arrow). Cells also contained a wild-type spoIIS locus with () or without () an additional copy of spoIISB at thrC.

	DISCUSSION

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Our results show that whenever its activity gets loose, SpoIISA induces striking abnormalities of the B. subtilis envelope and ultimately cell death. What makes SpoIISA a killer protein? Since it has structural features of an integral membrane protein, SpoIISA could act as a holin and allow some endolysin to gain access to the peptidoglycan (34). Local solubilization of the cell wall would obliterate its role as a protective barrier against osmotic pressure, leading to membrane disruption and consequently to the large plasmolysis zones observed by electron microscopy. It would also explain how SpoIISA toxic effects can spread to the mother cell in strains in which SpoIISA is synthesized exclusively in the forespore, since an endolysin would easily breach the thin septal cell wall separating the forespore from the mother cell. It is then intriguing that the spoIIS locus is located next to the PBSX prophage, immediately downstream of the xlyA gene encoding a phage muramidase (16). Yet, SpoIISA is not involved in PBSX-induced lysis since the presence of the xin15 mutation (that prevents induction of PBSX) does not suppress the sporulation defect of a spoIISB mutant nor the lethal effect of SpoIISA during exponential growth (data not shown).

However, SpoIISA does not show any similarity to known holins and is significantly larger than holins identified so far (34). It is therefore quite possible that the cytoplasmic membrane itself is the target of the toxic action of SpoIISA. For instance, SpoIISA could induce cell death directly by interfering with the respiration machinery whereas activation of autolysins and plasmolysis of the cytoplasmic membrane would be indirect consequences of the catastrophic failure of the dying cell. In this regard it should be noted that we have been unable to clone an intact spoIISA gene (without spoIISB) in E. coli, suggesting that SpoIISA is similarly toxic in E. coli (and that SpoIISB is similarly protective).

SpoIISB is the antidote neutralizing the killer protein SpoIISA. It is very likely that the two proteins interact directly and that the toxicity of SpoIISA (L103F) is due to the loss of that interaction. Several observations indicate that the relative levels of the two proteins are critical. For instance, the consequences of inducing SpoIISA synthesis during growth from the xylA promoter closely depend on the number of spoIISB genes in the cell. The presence of an internal promoter allowing sole transcription of spoIISB is a device that ensures an excess of SpoIISB molecules over SpoIISA, and it might be significant that this promoter is always at least threefold stronger than the promoter driving transcription of both spoIISA and spoIISB.

Complementation experiments strongly suggest that SpoIISA acts as an oligomer. On the one hand, the presence of wild-type SpoIISA makes SpoIISA(L103F) sensitive to SpoIISB provided that enough SpoIISB is supplied. On the other hand, SpoIISA(R38Q), which is apparently locked in an inactive conformation, prevents wild-type SpoIISA from releasing its activity in the absence of SpoIISB. In both cases the (partial) dominance of inactive SpoIISA is easily understood as a consequence of subunit mixing of a multimeric SpoIISA aggregate. It is then worth noting that such a molecular complex might be able to build a pore in the cytoplasmic membrane, a structural feature which could be the basis for the toxicity of SpoIISA.

Experiments in which SpoIISA was synthesized from the xylA or the spoIIQ promoters demonstrate that the vegetatively growing cell and the forespore are not immune to SpoIISA. Nevertheless, the absence of SpoIISB is cryptic during exponential growth when spoIISA is actively transcribed, and SpoIISA-induced death in stationary phase can be prevented by expressing spoIISB exclusively in the mother cell. Maybe some unidentified inhibitor restrains SpoIISA activity in growing cells and in the forespore of a spoIISB strain. Alternatively, SpoIISA might be intrinsically unstable and subjected to proteolysis but be stabilized in the mother cell. In both cases, synthesis of SpoIISA from a foreign promoter would override the mechanisms limiting its activity, with dramatic consequences for cell viability.

It is the deleterious action of SpoIISA on an essential function of the mother cell that prevents further morphological development and blocks the sporulation transcription program. The increase in ^F activity is probably the indirect consequence of the cells being stalled at stage II and the nonreplacement of ^F by ^G in the forespore. Deletion of the whole spoIIS locus has no effect on sporulation, and spoIIS is conspicuously absent from the genomes of all other sporulating gram-positive bacteria sequenced so far.

The genetic hierarchy between the two products of the spoIIS locus, one protein preventing the second one from hindering sporulation, has already been described for other pairs of proteins encoded by operons dispensable for sporulation. Such is the case for the antagonist of the starvation signaling pathway, the aspartate phosphatase RapA and its inhibitor, the imported peptide PhrA (25); for the repressor of some early sporulation genes, the DNA-binding protein Soj and its alternative partner, the chromosome partitioning protein Spo0J (5, 27); and for another repressor of early sporulation genes, the DNA-binding protein SinR and its inhibiting partner Sinl (2). Thus, a common gene organization of structurally and functionally unrelated sporulation regulatory circuits may be a general feature for B. subtilis genes encoding pairs of sporulation inhibitors and effectors.

Phenotypes formally similar to those of the spoIIS mutants have been reported for operons involved in bacterial cell death. Most of these operons are carried by plasmids and encode "addiction modules," a device that kills the cells having lost the plasmid (13). A few others are present on bacterial chromosomes and code for "antidote-toxin" pairs, whose activation in response to environmental signals may have a selective advantage for a subpopulation (4). It is all the more intriguing that the spoIIS products appear to be unique to B. subtilis, providing no clue to their evolutionary origin and their physiological role. Elucidating the latter will require identification of natural conditions in which SpoIISA activity is released.