INTRODUCTION

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Spore formation in Bacillus subtilis is a developmental pathway in which sequential, compartmentalized gene expression is achieved by interlocking cascades of regulatory factors and morphological cues (67). The primary environmental signal for initiation of sporulation is nutrient limitation (63), but this same condition also induces other adaptive responses (e.g., genetic competence, degradative enzyme synthesis, chemotaxis and motility, and antibiotic production) characteristic of slowly growing or stationary-phase cells.

The earliest morphological change that distinguishes a sporulating from a nonsporulating stationary-phase cell is the formation of an asymmetrically disposed division septum (48). During exponential growth, B. subtilis cells, like those of most other rod-shaped bacteria, divide exclusively at mid-cell. Mid-cell division requires the assembly at the septation site of a protein complex that includes FtsZ (4, 6), a tubulin-like GTPase (11, 50). Formation of a ring of FtsZ at the site of future septation is a prerequisite for association of other proteins with this site (25) and for septation itself. When a B. subtilis cell initiates sporulation, rings of FtsZ protein form at the two poles of the cell rather than at mid-cell (36). One of these rings becomes the site of asymmetric (polar) septation; the other ring dissociates. Bipolar localization of FtsZ is thought to be mediated by the product of a gene that depends on the Spo0A transcription factor for its expression, since in a spo0A mutant strain, polar rings of FtsZ do not form in stationary phase (36). Another factor required for polar septation is thought to be the product of a gene transcribed by the ^H form of RNA polymerase, since a spo0H (^H) mutant does not form polar septa even though FtsZ rings assemble at polar sites (36).

Asymmetric septation permits forespore-specific activation of ^F (1, 16, 42), the first step in a cascade of gene expression determined by sequentially active  factors (67). Soon after ^F becomes active, its activity leads to signals that activate ^E in the mother cell (27, 32, 37), followed by activation of ^G in the forespore (46, 68) and ^K in the mother cell (9, 10, 39).

The nutritional signal that initiates sporulation is unknown but is assumed to be created intracellularly by normal metabolism (63). It has long been known that the enzymes of the Krebs citric acid cycle are induced as cells leave exponential growth phase; activities of the enzymes are required for successful sporulation (18, 21, 54, 72). To investigate the specific roles of Krebs cycle enzymes in spore formation, we have analyzed the stages of sporulation blockage in mutants deficient in various steps of the cycle (8, 29, 31). We found that the absence of the third enzyme, isocitrate dehydrogenase (ICDH), causes a specific block at stage I (31); mutant cells enter stationary phase, organize their chromosomes in an axial filament (as in wild-type cells), and assemble apparently normal rings of FtsZ protein at both poles (31). No polar septation occurs, however. In the accompanying paper (41), we show that abnormally high accumulation of citrate is responsible, at least in part, for this phenotype.

To understand the basis for this stage I block and to identify proteins that may participate in or regulate asymmetric septation, we sought suppressor mutations that would restore polar septation to an ICDH (citC) mutant strain. To do so, we searched for spontaneous pseudorevertants of a citC null mutant in which expression of the ^F-dependent gene spoIIQ (38) was restored. One such mutation proved to be in the spoVG gene (53, 56, 58), whose product was not previously known to affect the pathway leading to asymmetric septation.

	MATERIALS AND METHODS

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Strains and growth conditions. The B. subtilis strains and plasmids used in this study are listed in Table 1. Cells were grown in nutrient broth sporulation (DS) medium (19) supplemented with antibiotics (5), if necessary. Sporulation was induced either by nutrient exhaustion in DS broth or by a resuspension method (65). LacZ indicator plates contained 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) at 80 µg/ml. Escherichia coli JM107 (71) was used for construction of plasmids.

DNA manipulation and transformation. Plasmid DNA was isolated from E. coli cells by a modified version of the method of He et al. (26). Other DNA manipulations were done by standard protocols (55). Preparation of electroporation-competent cells of E. coli and transformation with a Bio-Rad GenePulser apparatus (Bio-Rad Laboratories) were performed by the method of Dower et al. (14). Chromosomal DNA from B. subtilis was prepared as described previously (19). Transformation of B. subtilis cells with chromosomal DNA or plasmids was done by the procedure of Dubnau and Davidoff-Abelson (15).

Construction of an integrative genomic library of B. subtilis. A B. subtilis integrative plasmid, pPS34, and a B. subtilis genomic library were constructed by Pascale Serror (59). pPS34 was created by ligating the 2.8-kb SspI fragment of pBluescript SK() (pSK) (Stratagene, Inc.) to a 1.35-kb erythromycin resistance gene (erm) excised from pJPM9 (43) as a blunt-ended EcoRI-HindIII fragment. The erythromycin cassette was inserted in the same orientation as the lacZ gene of pSK. For construction of a B. subtilis genomic library, chromosomal DNA from strain FJS107 (Table 1) was digested separately with AluI, HinpI, and HpaII. After HinpI and HpaI fragments were blunt ended, they and the AluI fragments were inserted in separate ligations in the EcoRV site of pPS34. The cloned DNAs were pooled and, in a subsequent transformation, integrated into the chromosome of JH642 by homologous recombination. DNA from the pooled Erm^r transformants was prepared for use as a random insertion library.

Transfer of a suppressor mutation. Competent cells of strain SJB219 (citC::spc) (Table 1) were transformed with the chromosomal DNA from a spontaneous pseudorevertant, KMB77S3 (citC::spc amyE::[spoIIQ'-lacZ cat] spoVG2), and chloramphenicol-resistant (Cam^r) transformants were selected. Most were slightly blue on DS plates containing X-Gal, but some colonies were as dark blue as KMB77S3, indicating that the unlinked spoVG2 mutation and the spoIIQ'-lacZ fusion were cotransferred by congression. One of the congressants, KMB97, was used for subsequent mapping experiments (see Results).

Cloning of the spoVG2 mutant allele. The spoVG locus of KMB97, located within a 680-bp HindIII fragment, was amplified by PCR with the following primers: 5'-AAGTGATTCTGGGAGAGCCGGGATC-3' (which anneals about 170 bp upstream of the spoVG initiation codon) and 5'-AGGCTTACCGCAAACTGGATGAAGG-3' (which anneals about 280 bp downstream of the spoVG termination codon). The amplified DNA fragment (770 bp) was cloned directly into the modified EcoRV site of pT7Blue(R) (Novagen, Inc.). The resulting plasmid (pKM73) was sequenced to confirm that there were no additional mutations other than spoVG2 in the spoVG open reading frame. In order to eliminate possible PCR-derived mutations in the spoVG promoter, the BstXI-SacI fragment containing the spoVG2 mutation was excised and fused to the 5' end of wild-type spoVG in pKM70 which had been digested with the same enzymes, creating pKM89. To create pKM70, the spoVG-containing 680-bp HindIII fragment from pLS5 (a pBR322 derivative) (64) was inserted at the HindIII site of a version of pSK with a disrupted BstXI site in its multicloning site.

Overexpression of spoVG and preparation of anti-SpoVG antibodies. A strain producing a maltose-binding protein (MBP)-SpoVG fusion protein was constructed as follows. A BamHI site was introduced by PCR just upstream of the initiation codon of spoVG, and the amplified fragment was cloned in pT7Blue(R). A 300-bp BamHI fragment from the resulting plasmid was inserted in the proper orientation at the BamHI site of pMAL-c2 (New England Biolabs), a plasmid for construction of MBP fusions, to create pKM102. E. coli JM107 harboring pKM102 was used for overexpression of MBP-SpoVG.

Immunoblot analysis. Cells were harvested from a 50-ml culture in DS medium, washed once with 50 mM HEPES buffer (pH 8) containing 1 mM EDTA, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol, and 0.5 mM PMSF, and resuspended in 2 ml of the same buffer. Cells were disrupted by sonication followed by centrifugation to remove cell debris. Supernatant fluid (crude extract; 10 µg protein) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electrotransferred to an Immobilon-P (Millipore Corp.) membrane. The membrane was exposed to anti-SpoVG antibodies, and the immune complexes were visualized by using color-forming substrates of an alkaline phosphatase-linked secondary antibody (30).

Microscopic analysis. Fixation of cells and DNA staining with 4,6-diamidino-2-phenylindole (DAPI) were done essentially by the method of Wu et al. (70). Cells were harvested before and after induction of sporulation by the resuspension method (65) and fixed in 70% ethanol. Cell suspensions were kept at 4°C overnight, and subsequently subjected to nucleoid staining (1 µg of DAPI per ml). Samples were observed by using an Olympus BX60 microscope equipped with a C4742-95 digital camera (Hamamatsu). Images were processed with Image Pro Plus version 3.0 (Media Cybernetics) and Adobe Photoshop.

Construction of other plasmids. pKM56 was constructed by inserting the 680-bp HindIII fragment of pLS5 (see above) in the HindIII site of pDG1664, a B. subtilis integration vector for the thrC locus (22).

Other methods. DNA sequencing was done by using a Sequenase reagent kit (U.S. Biochemical Corp.) or by M. Berne, Tufts University Protein and Nucleic Acid Analysis Facility, using an ABI Automated Sequencer. Direct sequencing of PCR-amplified fragments was performed after treatment of the fragments with shrimp alkaline phosphatase and exonuclease I (U.S. Biochemical Corp.) according to the manufacturer's instructions. DNA sequences were analyzed with the DNA Strider and BLAST programs (2). -Galactosidase activity was measured and expressed as Miller units as described previously (61). All oligonucleotides used in this study were synthesized by M. Berne.

	RESULTS

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Isolation of spontaneous suppressor mutations that restore expression of spoIIQ-lacZ in a citC mutant. Sporulation of an ICDH (citC::spc) null mutant is blocked at stage I after expression of Spo0A-phosphate-dependent genes and bipolar localization of FtsZ rings early in stationary phase but before the formation of an asymmetric septum at one of the cell poles (31). Because of the failure of asymmetric septum formation, neither forespore-specific, ^F-dependent genes nor mother cell-specific, ^E-dependent genes are turned on in the citC mutant (31). To investigate the basis of the stage I block, extragenic suppressor mutations were sought by looking for spontaneous variants of the citC mutant in which expression of ^F-dependent genes was restored. For this purpose, we used a citC mutant strain (SJB295) carrying a fusion of the E. coli lacZ gene to the ^F-dependent promoter of the spoIIQ gene (38). In independent trials, two spontaneous mutants were isolated as dark blue colonies on plates of DS medium containing X-Gal. These colonies were as translucent as those of the citC mutant, indicating that they were still defective in sporulation. In transformation crosses, the suppressing mutations proved to be unlinked to citC or to the spoIIQ'-lacZ fusion integrated at the amyE locus. The spoIIQ'-lacZ fusion and the suppressing mutation of one of the strains were simultaneously moved by congression into strain SJB219 (citC::spc), creating strain KMB97 (see Materials and Methods). As shown in Fig. 1a, spoIIQ'-lacZ expression in strain KMB97 was increased in stationary phase to almost 50% of that of the wild-type strain.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Expression of spoIIQ'-lacZ (a) and spoIID'-lacZ (b) fusions in citC spoVG double mutants. Cells were grown in DS broth and harvested at the indicated times for measurement of -galactosidase activity. Symbols: , SJB294 (a) and SJB225 (b) (citC+); , SJB295 (a) and SJB229 (b) (citC); , KMB97 (citC spoVG2); , KMB198 (a) and KMB208 (b) (citC spoVG); , KMB200 (a) and KMB209 (b) (spoVG). Time zero is defined as the onset of stationary phase.

Identification of the spoVG2 mutation. To map the suppressing mutation in KMB97, we initially transformed strain KMB97 with a chromosomal library of random integrations of pPS34 (Erm^r; see Materials and Methods). DNA from the pooled transformants was then used to transform strain SJB295. Erm^r transformants that formed blue colonies on plates of DS medium containing X-Gal were tested individually for linkage of the suppressing mutation to erm. The location of an integrated plasmid showing about 50% linkage to the suppressing mutation was determined by cleavage of chromosomal DNA with EcoRI, ligation at a low DNA concentration, transformation of E. coli, extraction of plasmid, and sequencing of the cloned insert. The site of plasmid insertion was in the 5' end of the mfd gene (44), located at 7° on the chromosomal map. Further genetic crosses demonstrated that the mutation is 30% linked to abrB (47), located about 15 kb upstream of the mfd gene, and 70% linked to each of two insertion mutations previously shown to flank the spoVG gene (24). The spoVG locus of KMB97 was amplified by PCR and direct sequence analysis of the PCR-amplified fragments showed a point mutation (CCT to CTT), causing a single amino acid substitution from proline to leucine at residue 63. This mutation was designated spoVG2. Subsequently, the spoVG2 mutant allele was cloned and the presence of the mutation was confirmed by sequence analysis. The second, independently isolated, suppressor mutant was found to have the same mutation, perhaps because this mutation causes the same phenotype as a null mutation (see below).

Proof that spoVG2 is the suppressing mutation and is recessive. When the wild-type spoVG gene was introduced at the thrC locus in KMB97, the suppressor mutant phenotype disappeared (Fig. 2), indicating that spoVG2 is required for suppression and that the spoVG2 mutation is recessive to the wild-type spoVG⁺. The recessiveness of spoVG2 suggests that it is a loss-of-function mutation. To confirm this point, we tested the ability of null alleles of spoVG to suppress the citC mutation. When spoVG::Tn917HU265 (56) or spoVG::tet (66) was introduced into the citC mutant (creating strain KMB136 or KMB198, respectively), the expression of the spoIIQ gene in each of the citC spoVG mutants was indistinguishable from that in KMB97 (Fig. 1a and data not shown), implying that the spoVG2 mutation abolishes a negative effect of SpoVG on ^F activation in the citC mutant.

View larger version (22K): [in this window] [in a new window]   FIG. 2.   spoVG2 is a recessive mutation. Cells carrying a spoIIQ'-lacZ fusion were grown in DS broth and harvested at the indicated times for measurement of -galactosidase activity. Symbols: , SJB294 (citC+); , SJB295 (citC); , KMB97 (citC spoVG2); , KMB158 (citC spoVG2 thrC::spoVG+). Time zero is defined as the onset of stationary phase.

Expression of spoVG in a citC mutant. Although citC mutant cells were known to express the spoVG gene and have no defect in ^H activity (31), we reexamined spoVG gene expression in detail. As shown in Fig. 3a, spoVG expression was induced at the normal time in the mutant at the onset of sporulation but, unlike the case for the wild-type strain, failed to shut off after T₁ (1 h after the onset of stationary phase), reaching a maximum level approximately twofold higher than that seen in the wild-type strain.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Expression of a spoVG'-lacZ fusion in a citC mutant. (a) Wild-type (SJB78; ) and citC mutant (KMB162; ) strains were grown in DS broth and harvested at the indicated times for measurement of -galactosidase activity. (b) Samples of wild-type (SJB294) and citC mutant (SJB295) cultures were harvested at the 1-h intervals after the onset of stationary phase indicated over the gels and disrupted by sonication. Proteins (10 µg) in the crude extract were separated in sodium dodecyl sulfate-polyacrylamide gels and subjected to immunoblot analysis with anti-SpoVG antibodies.

Overexpression of spoVG in B. subtilis. To assess further the potential negative effect of SpoVG on asymmetric septation, we measured the effect on wild-type cells of overexpression of spoVG. We used a low-copy-number plasmid, pHP13, for this purpose, since it is known that high dosage of the ^H-dependent spoVG promoter results in severe inhibition of sporulation at an early stage (3, 73). Figure 4 shows that, in wild-type cells harboring spoVG⁺ on pHP13 (pKM98), ^F-dependent spoIIQ expression was significantly delayed. This inhibitory effect was not caused by titration by the spoVG promoter of any regulatory factor, since no detectable difference in spoIIQ expression was found in cells carrying the plasmid-borne spoVG2 allele compared to cells carrying the vector only.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Expression of spoIIQ'-lacZ in strains harboring the spoVG gene and the spoVG2 allele on a multicopy plasmid. Cells having pHP13 (vector only; ), pKM98 (spoVG+; ), or pKM157 (spoVG2; ) were grown in DS broth and harvested at the indicated times for measurement of -galactosidase activity. Time zero is defined as the onset of stationary phase.

Sporulation gene expression in a citC spoVG double mutant. Since colonies of a citC::spc spoVG::tet double mutant were as translucent as those of the original citC mutant and the number of heat-resistant spores, measured at about T₂₀, was nearly identical to that of the citC mutant (Table 2), we examined later stages of the sporulation pathway to identify the defect. The expression of the mother cell-specific, ^E-dependent spoIID gene (52) is normally induced following activation of ^F in the forespore. However, in the citC spoVG double mutant, there was relatively little induction of spoIID'-lacZ (Fig. 1b). As expected from the low level of ^E activity in the mother cell, there was little or no expression of the later sporulation genes sspE (17) and cotA (57), which are transcribed by RNA polymerases containing ^G and ^K, respectively (data not shown). These defects were not due to the spoVG mutation, since a spoVG single mutant had no detectable deficit in spo gene expression (data not shown) and sporulated at close to the wild-type frequency (reference 53 and Table 2).

citC spoVG

View larger version (188K): [in this window] [in a new window]   FIG. 5.   Thin-section electron microscopic analysis. A sample of a culture of citC spoVG mutant cells (KMB197) was harvested at T6 and prepared for transmission electron microscopy as described in Materials and Methods.

Minicell formation in spoVG null mutant. Since the phenotype of the spoVG mutant suggests that the wild-type protein interferes with asymmetric septation, we tested the effects on cell division and sporulation of a spoVG mutation alone or in combination with a mutation in minD, a gene whose product inhibits polar septation during exponential growth (34, 35, 69). We confirmed that single null mutations in either spoVG or minD had relatively small effects on sporulation frequency (34, 35, 53). In our hands, the numbers of viable cells and spores obtained with either strain were decreased to about 50% of those of the wild-type strain after growth overnight in DS broth (Table 2 and data not shown). The minD spoVG double mutant suffered further losses in viability and spore titer to 30 and 10%, respectively (data not shown). The impaired ability of the double mutant to maintain viability and produce spores was reflected in the production of minicells by single and double mutants. O. Resnekov (51) had noted previously that a spoVG null mutant produces minicells. We estimated the minicell fraction at 2 to 3% of total spoVG mutant cells during vegetative growth (Fig. 6 and Table 4). A minD mutant produced about 13% minicells.

View larger version (73K): [in this window] [in a new window]   FIG. 6.   Minicell formation in a spoVG mutant. Cells of strain KMB200 (spoVG) were grown to mid-exponential phase and induced to sporulate by the resuspension method of Sterlini and Mandelstam (65). Ethanol-fixed cells were observed under phase-contrast microscopy (left-hand panels), and nucleoids were visualized by DAPI staining (right-hand panels). (a to c) Exponential-phase cells, showing an anucleate minicell (a), a minicell containing a condensed nucleoid (b), and a disporic cell (c). (d) Cell population of the mutant at 1 h after resuspension. Arrows 1 to 4 indicate anucleate minicells, arrow 5 indicates a sporulating cell at stage 0-I, arrow 6 indicates a sporulating cell at stage II, and arrows 7 and 8 indicate disporic cells.

View this table: [in this window] [in a new window]   TABLE 4.   Minicell formation in spoVG mutant

	DISCUSSION

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Null mutations in the spoVG gene partially relieve the stage I block of a citC mutant, allowing a significant fraction of the citC mutant cell population to form an asymmetric septum and activate expression of ^F-dependent genes in the forespore compartment. This evidence suggests that a normal function of ICDH overcomes the activity of SpoVG, which is itself an inhibitor or a negative regulator of the ^H-dependent pathway to the asymmetric septum. In other work, we have shown that it is the enzymatic activity of ICDH that is critical for completing stage I (31, 41).

spoVG was the first developmentally regulated gene cloned from B. subtilis (58). Close homologs are now known to exist in Bacillus megaterium, Clostridium acetobutylicum, Borrelia burgdorferi, and Archaeoglobus fulgidus, organisms which collectively represent gram-positive and -negative eubacteria and the archaea. Proline-63, the residue mutated in our suppressor strains, is conserved in all five SpoVG proteins.

The absence of SpoVG in sporulating cells of B. subtilis is known to cause aberrations in the germ cell wall and the cortex, defects seen at T₅ to T₆ (stage V) (53), and to make the stage II defect of a spoIIB mutant more severe (40). However, extensive gene expression studies have shown that the spoVG gene is a very early stationary phase gene, expressed at a significant level even during exponential growth phase and further induced at the end of exponential phase (45, 74). This transcription depends on the ^H form of RNA polymerase and is partially repressed during early exponential phase by a global negative regulator, AbrB (74). The concentration of SpoVG protein reaches a maximum level at the onset of stationary phase and decreases substantially after T₂, indicating that SpoVG most likely has its primary function early in stationary phase. In the citC mutant, spoVG expression is induced at the normal time but continues to increase even after several hours in stationary phase. Moreover, the level of SpoVG protein remains high at a time when it is greatly diminished in wild-type cells. Thus, the dependence of asymmetric septation on ICDH activity might be attributed to an effect on SpoVG synthesis or stability. Asymmetric septation, however, is an early event in stationary phase; it is uncertain whether prolonged expression of SpoVG (seen after T₁) is in fact responsible for the primary effect of the citC mutation. On the other hand, overexpression of spoVG in wild-type cells interferes with asymmetric septation.

The MinCD complex is a cell division inhibitor in exponential-phase cells of B. subtilis (34, 35, 69), as well as in E. coli (12, 13). Cells lacking these proteins form septa at potential division sites near cell poles and produce anucleate minicells during vegetative growth (13). The Min proteins appear to mask the polar cell division sites in normal growing cells, restricting FtsZ ring formation to medial sites (49). (Under some growth conditions, other proteins can carry out this function in B. subtilis [36a].) The masked sites, however, must become accessible to FtsZ when B. subtilis cells enter the spore formation program, implying that polar septation inhibitors (e.g., MinCD) lose activity in stationary phase. Our finding that deletion of the spoVG gene increases the rate and extent of asymmetric septation in both wild-type and citC mutant cells suggests that a normal role of SpoVG in early stationary phase is to temporarily prevent asymmetric septation after the Min proteins cease to function. Perhaps the subtle defects observed in late sporulating cells of a spoVG mutant (40, 53) are due to the loss of precise timing of asymmetric septum formation.

Although the primary function of SpoVG seems to be to regulate asymmetric septation in stationary-phase cells, the spoVG gene is also expressed, albeit at a lower level, in a population of exponential-phase cells. The fact that an exponential-phase culture of the spoVG mutant contains some minicells with condensed nucleoids and cells with a disporic phenotype is consistent with a heterogeneous population in which some cells initiate sporulation while others grow. This supposition is consistent with our finding that appearance of the aberrant cells is dependent on the Spo0A phosphorelay.

While inactivation of SpoVG partially overcomes the stage I block of a citC mutant, the cells do not complete the sporulation process or even proceed beyond stage III. Moreover, only a subpopulation (<30%) of the citC spoVG double mutant cells form asymmetric septa and the level of ^F-dependent gene expression reaches only 30 to 50% of the level in wild-type cells. Further suppression of the stage I block in a citC mutant requires reducing citrate synthesis or raising the pH of the medium and supplementation with MnCl₂ to compensate for overaccumulation of citrate (41).

The mechanism by which SpoVG regulates the extent and timing of asymmetric septation is unknown. SpoVG may interact directly with the septation apparatus or may act indirectly by regulating the synthesis or stability of proteins required for septation. Future experiments will be directed toward identifying the mechanism by which SpoVG regulates asymmetric septation and the means by which ICDH activity intervenes in this process. A working model has the following elements. As cells make the transition from exponential growth to stationary phase, citC and spoVG are simultaneously induced. At the same time, polar sites for assembly of FtsZ-containing cell division complexes become unmasked, but polar septation is still prevented in an unknown way by newly accumulated SpoVG. As Krebs cycle enzymes, including ICDH, begin to function, acidic metabolites of the glycolytic pathway are assimilated, raising extracellular pH, inactivating SpoVG, and inducing asymmetric septation, a process that requires one or more Mn²⁺-dependent proteins. Our data, however, do not rule out the possibility that low pH, Mn²⁺ limitation, and SpoVG affect polar septation by independent mechanisms.