Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Abstract

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.

Penicillin-binding proteins (PBPs) are essential for the synthesis of cell wall peptidoglycan (PG) in bacteria (reviewed in reference 12). In Escherichia coli, three different classes of PBPs have been identified. Class A high-molecular-weight (HMW) PBPs possess both transglycosylase activity, which is needed for PG strand polymerization, and transpeptidase activity, which is required for cross-linking of peptide side chains on PG strands (18, 28). Class B HMW PBPs exhibit transpeptidase activity (17). Low-molecular-weight PBPs are d-ala–d-ala carboxy- or endopeptidases whose activity contributes to the regulation of the degree of PG strand cross-linking (2, 21, 40).

PBPs of these three classes are also present in Bacillus subtilis, in which they are essential for PG synthesis during vegetative growth, sporulation, and spore outgrowth (reviewed in reference 6); the genes encoding 10 of the major PBPs in vegetative and sporulating cells have now been identified (7, 8, 26, 27, 34–37, 48, 52). Thus far, only the class B HMW PBP2b, encoded by pbpB, has been found to be essential for cell growth, most likely due to the role of this PBP in septum formation during vegetative growth (52). The majority of other B. subtilis PBPs appear to carry out redundant reactions, since the loss of any one of many PBPs has little or no effect on cell growth, morphology, sporulation, or spore properties (27, 35–37). However, the loss of one of several other PBPs does result in a notable phenotype. Examples of these phenotypes include the following: (i) the loss of the sporulation-specific class B HMW PBP SpoVD blocks sporulation, probably because of the requirement of this protein for the synthesis of the spore cortex (8); (ii) the loss of the sporulation-specific low-molecular-weight PBP5*, encoded by dacB, results in reduced heat resistance of spores and an altered spore cortex structure, suggesting a role for this protein in cortex maturation (1, 5, 32, 33); (iii) the loss of the HMW class B PBP2a, encoded by pbpA, results in the initial generation of large spherical cells during spore outgrowth (26); and (iv) the loss of the HMW class A PBP1, encoded by ponA, causes slight reductions in cell diameter and growth rate in rich media, as well as slight cell bending and decreased sporulation efficiency (4, 34, 38). Interestingly, B. subtilis strains lacking both PBP1 and other class A HMW PBPs are viable and exhibit only slight changes in their growth rates and cell morphology compared to a strain lacking only PBP1 (38).

B. subtilis has three genes encoding known class A HMW PBPs (ponA, pbpD, and pbpF) and one pbp gene, ywhE, encoding a putative class A HMW PBP (22, 34, 36, 37), while E. coli has only three genes encoding class A PBPs, ponA, ponB, and pbpC (15). In E. coli, the loss of ponA, encoding PBP1A, is not accompanied by significant changes in either cell morphology or growth rate, and the loss of ponB, encoding PBP1B, results in only a slightly reduced growth rate. However, the disruption of both genes is lethal (19, 53). Disruption of a single ponA homolog in Neisseria gonorrhoeae is also lethal (41). The lack of a more dramatic phenotype in B. subtilis ponA mutants and mutants lacking multiple class A HMW PBPs was therefore surprising given the proposed importance of PBP1 and class A HMW PBPs in PG synthesis.

As noted above, the growth rate of a B. subtilis ponA mutant in a rich medium (2×YT) was slightly lower than that of the wild-type strain (38). However, the difference between the growth rates of these two strains in a second rich medium (2×SG) was smaller (38). While there are a number of differences between these two media, one substantial difference is in their levels of divalent cations, since 2×SG has about fivefold-higher levels of both Mg²⁺ and Ca²⁺. Divalent cations such as Mg²⁺ are essential for bacterial growth, and in both E. coli and B. subtilis, low Mg²⁺ levels cause decreases in vegetative-growth rates (24, 51). Low levels of Mg²⁺ have also been shown to cause changes in cell morphology in a number of different bacterial species, including B. subtilis (43, 51). Thus, it is possible that the loss of PBP1 increases the divalent-cation requirement for normal growth of B. subtilis.

In this work, we demonstrated that B. subtilis mutants lacking PBP1 do indeed have an increased requirement for Mg²⁺ or Ca²⁺ during vegetative growth and that growth of ponA mutants in media with a low levels of divalent cations gives rise to an altered cell morphology. Outgrowing spores lacking PBP1 also exhibit distinct morphological changes in a medium with low levels of divalent cations. This work suggests that while B. subtilis class A HMW PBPs carry out redundant functions in rich media, PBP1 is uniquely required for cell growth when levels of divalent cations are low.

MATERIALS AND METHODS

Strains used, vegetative growth, sporulation, and spore germination and outgrowth.

The B. subtilis strains used in this work are listed in Table 1; all strains were derived from PS832, a prototrophic revertant of strain 168. In strain PS2062, a spectinomycin cassette has replaced ∼60% of the ponA coding region, including the entire N-terminal domain and half of the C-terminal domain of PBP1, including the active-site serine (34). B. subtilis was routinely grown and sporulated at 37°C in antibiotic-free 2×SG medium (23). Spores were then purified by repeated washing in water as described previously (30). After a 30-min heat shock treatment in water at 70°C, purified spores were inoculated to an initial optical density at 600 nm (OD₆₀₀) of 0.3 to 0.5 in various media (see below) containing 4 mM l-alanine for germination at 37°C (30). In some experiments, 30 min after the initiation of spore germination, spores were centrifuged at 3,000 × g for 5 min at room temperature to remove molecules released during the initial stages of germination; the pellet fraction was then resuspended in an equal volume of fresh medium. In other experiments, dormant spores were decoated with a solution containing 0.5% sodium dodecyl sulfate, 0.1 M dithiothreitol, 0.1 M NaCl, and 0.1 M NaOH at 65°C for 30 min (50), washed 10 times with water, resuspended in water, and heat shocked as described above.

TABLE 1.

B. subtilis strains used in this study

Strain	Relevant genotypea	Source or reference
PS832	Prototrophic revertant of strain 168	Laboratory stock
PS1869	ΔpbpF MLSr	36
PS2022	ΔpbpD MLSr	37
PS2062	ΔponA Spr	34
PS2156	pbpF::Cmr ΔponA Spr	38
PS2182	ΔpbpD MLSr ΔponA Spr	38
PS2352	ΔpbpC Cmr	27
PS2363	ΔponA Spr ΔpbpC Cmr	27

^aAbbreviations: Cm^r, chloramphenicol resistant; MLS^r, erythromycin and lincomycin resistant; Sp^r, spectinomycin resistant. 

The media used for vegetative growth and spore germination and outgrowth were the following: 2×YT (38) (total Mg²⁺ and Ca²⁺ concentrations of 420 and 85 μM, respectively [Difco typical analysis of medium components]), 1× Penassay broth (PAB; Difco) (total Mg²⁺ and Ca²⁺ concentrations of 210 and 40 μM, respectively), a modification of Spizizen’s modified minimal medium containing 0.1% Casamino Acids and no added Mg²⁺ (SMMM) (45) (total Ca²⁺ concentration of 100 μM), and 2×SG (total Mg²⁺ and Ca²⁺ concentrations of 2.6 and 1.1 mM, respectively). Note that the concentrations of Mg²⁺ and Ca²⁺ in these media are the total amounts, not the free-cation concentrations. The OD₆₀₀ values of all cultures was monitored with a Genesys model 5 spectrophotometer. For vegetative growth in PAB medium, cells were grown overnight on plates of 2×SG containing the appropriate antibiotics, resuspended in PAB medium, and inoculated into liquid PAB medium to give an initial OD₆₀₀ of 0.01 to 0.05. To test the cation requirements of various B. subtilis strains, they were grown in PAB medium with additions as noted in individual experiments.

Cell and spore fixation, microscopy, statistical analysis, and cell wall staining.

Vegetative cells and outgrowing spores were harvested by centrifugation, fixed in 2.5% glutaraldehyde, and rinsed in phosphate-buffered saline (PBS) as previously described (26). The fixed samples were placed on coverslips coated with 0.01% polylysine, prepared for light microscopy, and visualized by differential interference contrast (DIC) microscopy, using a Noran confocal laser scanning microscope equipped with a 100× Plan-APO chromatic oil immersion lens (Zeiss) as described previously (26).

When determining the percentage of cells exhibiting morphological changes, at least 150 cells were examined by phase-contrast microscopy. A cell was considered bent when its poles were not at a 180° angle; in this analysis, we excluded cells with a septum at the apex of the angle. A filament was defined as any cell exceeding two cell lengths. Cell wall was labeled with wheat germ agglutinin (WGA) conjugated to Oregon Green (Molecular Probes) (31), and DNA was stained with 4,6-diamidino-2-phenylindole (DAPI). Fixed cells on polylysine-coated coverslips were treated with lysozyme (1 mg/ml) for 30 s, rinsed three times in PBS, blocked for 10 min with 2% bovine serum albumin in PBS, and incubated with a solution consisting of 5 μg of WGA and 1.25 μg of DAPI per ml for 90 min. The cells were rinsed eight times with PBS, placed on coverslips by the use of a SlowFade antifade kit (Molecular Probes), and viewed with the Noran confocal laser scanning microscope as described above but with a fluorescein filter.

Wild-type and ponA spores undergoing outgrowth in PAB medium plus 500 mM NaCl were harvested by centrifugation 130 min after the initiation of spore germination, fixed, processed, and analyzed by transmission electron microscopy as described previously (42).

RESULTS

Vegetative growth of strains lacking HMW PBPs.

Previous work has shown that strains lacking single HMW PBPs grow in both rich (2×YT and 2×SG) and minimal (Spizizen’s [45]) media, although a ponA strain lacking PBP1 grows slower than the wild-type strain (34, 38). Interestingly, the growth rate of the ponA strain was reduced the most (about 2-fold) in 2×YT medium and significantly less (1.3- to 1.4-fold) in another rich medium (2×SG) and a minimal medium (38). One significant difference between these media is that the concentration of total Mg²⁺ and Ca²⁺ is two- to sixfold lower in 2×YT medium than in 2×SG or minimal medium.

To directly assess the importance of divalent cations for vegetative growth of cells lacking HMW PBPs, we used PAB medium, which has even a lower level of total Ca²⁺ plus Mg²⁺ (about 250 μM) than 2×YT medium (which has about 500 μM). While B. subtilis strains lacking HMW PBP2a, -2c, -3, or -4 grew as well as the wild-type strain in PAB medium, the strain lacking PBP1 exhibited little if any growth (Fig. 1 and data not shown). The growth rate of ponA cells in PAB medium varied between none and very slow depending on the lot of PAB medium used, probably due to differences in composition or strength between lots.

FIG. 1.

Effect of different Mg²⁺ concentrations on growth of ponA cells. Cells were grown at 37°C in PAB medium with addition of MgCl₂ at various concentrations. The symbols for the strains and MgCl₂ concentrations added are as follows: □, PS832 (wild type), no additions; ○, PS2062 (ponA), no additions; •, PS2062 (ponA), 50 μM Mg²⁺; ◊, PS2062 (ponA), 100 μM Mg²⁺; ▵, PS2062 (ponA), 1 mM Mg²⁺; and ■, PS2062 (ponA), 10 mM Mg²⁺.

To confirm that the inability of ponA cells to grow in PAB medium was due to low levels of divalent cations, a variety of divalent cations were added in attempts to restore growth. Strikingly, addition of 500 μM MgCl₂, MgSO₄, or CaCl₂ was sufficient to restore rapid growth of the ponA mutant, while addition of 500 μM BaCl₂, CoCl₂, or MnCl₂ was not (Fig. 1 and data not shown). Restoration of growth of ponA mutants in PAB medium by Mg²⁺ or Ca²⁺ was concentration dependent, and the addition of as little as 50 μM MgCl₂ was sufficient to allow significant growth (Fig. 1). Addition of 50 μM Mg²⁺ to SMMM also allowed rapid growth of wild-type cells (doubling time, 35 min) but only slow growth of ponA cells (doubling time, 120 min). Furthermore, the wild-type strain, when incubated in SMMM for 90 min (during which time no growth occurred), was able to initiate growth when the MgCl₂ concentration of the culture was subsequently brought to 50 μM, while the ponA strain was not (data not shown). The growth of double mutants, lacking PBP1 and either PBP2c, -3, or -4, in liquid PAB medium with or without added Mg²⁺ was similar to that of the strain lacking only PBP1 (Table 2). In contrast to the results with PAB medium, all PBP mutants examined in this work grew in 2×YT medium, although those lacking PBP1 grew more slowly than the wild-type strain, as previously reported (data not shown) (34).

TABLE 2.

Phenotypes of HMW PBP mutants in media with low levels of divalent cations (PAB ± 50 μM MgCl₂)

Strain (relevant genotype)	Vegetative growth ratea	Vegetative-cell morphologya,b	Spore outgrowth kineticsa	Outgrowing-spore morphologya,b
PS832 (wtc)	wt	Straight rods	wt	Some (∼11%) bent rods
PS2062 (ponA)	None	Significant numbers of bent (23%) and filamentous (37%) cells	Delayed compared to wild type	Many (∼62%) bent cells
PS2352 (pbpC)	Like wt	Like wt	Like wt	Like wt
PS2022 (pbpD)	Like wt	Like wt	Like wt	Like wt
PS1869 (pbpF)	Like wt	Like wt	Like wt	Like wt
PS2363 (ponA pbpC)	Like ponAd	Like ponAd	Like ponAd	Like ponAd
PS2182 (ponA pbpD)	Like ponAd	Like ponAd	Slightly slower than ponA	Many (72%) extremely bent and curled cellse
PS2156 (ponA pbpF)	Like ponAd	Like ponAd	Like ponAd	Like ponA

^aGrowth and spore outgrowth of the wild-type strains was in PAB medium. 

^bGrowth of ponA strains was in PAB medium with 50 μM MgCl₂ (Fig. 1), while spore outgrowth took place in PAB medium without additions (see text). Cells were harvested, fixed, and examined by phase-contrast microscopy as described in Materials and Methods. Examples of vegetative-cell morphology are shown in Fig. 2, while morphologies of outgrowing spores are shown in Fig. 4. Values in parentheses are the percentages of cells or outgrowing spores displaying the described morphology. 

^cwt, wild type. 

^dCharacteristics were essentially identical to those of strain PS2062 (ponA). 

^eOutgrowth in 2×YT medium results in formation of helical cells (Fig. 4G). 

Previous work showed that some of the ponA cells grown in 2×SG medium were bent, in contrast to wild-type cells and those of other single PBP mutants, which were not bent (38). Analysis by DIC microscopy showed that a large fraction of ponA cells grown at a low [Mg²⁺] (50 μM added to PAB medium) were significantly bent and grew as filaments, in contrast to the absence (<1%) of these types of cells in wild-type cultures grown in PAB medium (Fig. 2A and B; Table 2). Microscopic analysis of ponA cells colabeled with WGA conjugated to Oregon Green (to stain cell wall and septa) and to DAPI (to stain DNA) showed that while some filamentous cells had regularly placed septa, many had very few or no septa (Fig. 2D and data not shown); this was also confirmed by staining the cytoplasm with propidium iodide (data not shown). However, nucleoids were regularly spaced throughout all filaments examined (Fig. 2E and data not shown). In PAB medium, strains lacking PBP2c, -3, or -4 looked like wild-type cells, while strains lacking PBP1 and either PBP2c, -3, or -4 looked like the strain lacking only PBP1 (Table 2). Strikingly, the addition of 10 mM MgCl₂ to PAB medium eliminated the bending and filamentation of ponA cells, although their reduced diameter, observed previously, remained (data not shown). The rodB1 mutation causes both a reduced growth rate and the formation of spherical cells in B. subtilis, and the effects of this mutation are suppressed by addition of either Mg²⁺ or 10 mM NaCl (39). However, neither NaCl nor KCl at a concentration of 10, 100, or 500 mM was able to restore growth of the ponA strain in PAB medium (data not shown).

FIG. 2.

Morphology of ponA cells grown in PAB medium with 50 μM Mg²⁺ (A) and of wild-type cells grown in PAB medium without additions (B) or with 500 mM NaCl (C). ponA and wild-type cells were inoculated to an OD₆₀₀ of 0.02 in PAB medium and harvested after 90 min, while wild-type cells grown in PAB medium plus 500 mM NaCl were inoculated to an OD₆₀₀ of 0.1 and harvested after 120 min. Cells were fixed, prepared for microscopy, and viewed as described in Materials and Methods. The arrows in panel C show cells with altered morphology. ponA filaments from the same culture as that shown in panel A were stained for cell wall and septa with WGA (D) and for DNA with DAPI (E) as described in Materials and Methods. The arrow in panel D shows a septum. Bars, 10 μm.

When wild-type cells were inoculated into PAB medium with 500 mM NaCl to an initial OD₆₀₀ of 0.1, they initiated growth following a lag. The OD₆₀₀ had increased to between 0.2 and 0.3 by 120 min, at which time it dropped dramatically (data not shown). This latter change was accompanied by drastic changes in cell morphology, including the loss of normal rod shape, swelling of parts of individual cells, and cell lysis (Fig. 2C). Since ponA cells failed to grow in PAB medium with 500 mM NaCl, the morphological changes seen with the wild-type strain in this medium were not observed (data not shown). Supplementation of PAB medium containing 500 mM NaCl with 10 mM Mg²⁺ allowed the growth of and restored the rod-shaped morphology to both wild-type and ponA cells (data not shown). The effects of NaCl on wild-type cell growth and morphology do not appear to be due to changes in the osmolarity of the growth medium, since wild-type cells grew normally, albeit only after a lag, in PAB medium with 1.0 M sorbitol while ponA cells did not grow; however, ponA cells grew normally in PAB medium with 1.0 M sorbitol upon addition of 10 mM Mg²⁺ (data not shown).

Outgrowth of spores lacking HMW PBPs.

Previous work has shown that ponA spores proceed through germination and outgrowth relatively normally in 2×YT medium (34). However, since ponA is transcribed, and PBP1 is present very shortly after the initiation of spore germination (29, 34), we examined whether elevated levels of divalent cations were also essential to ponA spores during germination and outgrowth. Initial experiments showed that spores lacking only PBP1 or PBP1 and either PBP2c, -3, or -4 initiated spore germination essentially identically to wild-type spores in PAB medium, as measured by the initial drop in OD₆₀₀ (Fig. 3A and data not shown). Given the requirement of increased Mg²⁺ for vegetative growth of a ponA strain, it was surprising that ponA spores did undergo outgrowth in PAB medium, although the process was delayed relative to that of wild-type spores (Fig. 3A). One possible explanation for the eventual outgrowth of ponA spores is the spore’s release of large amounts of divalent cations during germination (13). We attempted to remove these molecules by centrifugation of spores and resuspension of the pellet in fresh medium 30 min after initiating spore germination. While this did not significantly alter the outgrowth kinetics of ponA spores (data not shown), it is possible that the asynchrony of spore germination and/or the binding of divalent cations by the spore coats and exosporium precluded removal of sufficient divalent cations by centrifugation. Indeed, while decoated ponA spores initiated germination in PAB medium normally, spore outgrowth was extremely delayed and very little cell elongation occurred (Fig. 3A). In contrast, decoated wild-type spores were able to undergo outgrowth and elongation in PAB medium, albeit after a slight lag (Fig. 3A). Since the addition of 10 mM Mg²⁺ to PAB medium was sufficient to restore outgrowth to decoated ponA spores (data not shown), this suggests that coat proteins may have indeed retained sufficient Mg²⁺ and Ca²⁺ to allow spore outgrowth and subsequent cell growth in divalent-cation-deficient PAB medium. Additional mutations eliminating PBP2c or PBP3 in a ponA background did not further delay spore outgrowth in PAB medium; however, the loss of both PBP1 and PBP4 did (Table 2) (see below). The kinetics of spore outgrowth for all pbp mutant strains became similar to that of wild-type spores when 10 mM Mg²⁺ was included in the PAB medium (data not shown).

FIG. 3.

Kinetics of germination and outgrowth of spores of HMW PBP mutants in PAB medium without additions (A), with 500 mM NaCl (B), and with both 500 mM NaCl and 10 mM Mg²⁺ (C). Spores were heat shocked and germinated as described in Materials and Methods. Note the different scales on the vertical axes. The symbols for the strains and their genotypes are as follows: □, PS832 (wild type); ■, PS832 (wild type), decoated; ○, PS2062 (ponA); •, PS2062 (ponA), decoated; ◊, PS2022 (pbpD); and ▵, PS1869 (pbpF).

Microscopic examination of wild-type spores 120 min after the initiation of spore germination in PAB medium revealed that a significant number of cells were slightly bent (Fig. 4A; Table 2); outgrowth of ponA spores resulted in an even higher percentage of severely bent cells (Fig. 4B; Table 2). The percentage of bent cells dropped from 62% after 120 min to 27% by 180 min after the initiation of ponA spore germination, but a significant percentage of filaments was observed at the later time (14%; n = 207). While outgrowth of mutant spores lacking PBP1 and either PBP2c or PBP3 appeared similar to that of ponA spores, outgrowing cultures of spores lacking PBP1 and PBP4 contained a large percentage of forms that were extremely bent and curled (Fig. 4C; Table 2). As noted above for the effect of Mg²⁺ on rates of spore outgrowth, the addition of high levels of Mg²⁺ to PAB medium also suppressed most of the cell bending associated with outgrowth of spores lacking PBP1, with or without additional pbp mutations (data not shown) (see below).

FIG. 4.

Morphology of outgrowing spores of the wild type and of HMW PBP mutants. Spores were heat shocked, and spore germination and outgrowth were accomplished as described in Materials and Methods. Outgrowing spores were harvested by centrifugation 120 min after the initiation of spore germination (unless otherwise noted), fixed, prepared for microscopy, and examined as described in Materials and Methods. The strains and the outgrowth conditions were as follows: (A) PS832 (wild type), PAB medium with no additions; (B) PS2062 (ponA), PAB medium with no additions; (C) PS2182 (ponA pbpD), PAB medium with no additions; (D) PS832 (wild type), PAB medium plus 500 mM NaCl, harvested after 180 min (note the presence of lysed cells); (E) PS2062 (ponA), PAB medium plus 500 mM NaCl, harvested after 180 min; (F) PS2062 (ponA), PAB medium plus 500 mM NaCl and 10 mM Mg²⁺, harvested after 180 min; and (G) PS2182 (ponA pbpD), 2×YT medium with no additions, harvested after 150 min. The round spots visible in some fields are the result of dust in the camera. Bars, 10 μm.

As was observed for cell growth, germination and outgrowth of wild-type spores and spores lacking either PBP2c, -3, or -4 were similar for 120 to 150 min in PAB medium plus 500 mM NaCl (Fig. 3B and data not shown) but slower than for wild-type spores in PAB medium alone (compare Fig. 3A and B). Cell elongation was also slower during spore outgrowth in PAB medium with 500 mM NaCl than in PAB medium alone. In addition, shortly after cell elongation initiated in PAB medium with 500 mM NaCl, the OD₆₀₀ of the culture dropped dramatically (Fig. 3B). Microscopic examination of cultures at this time showed that the drop in OD₆₀₀ was accompanied by the formation of dumbbell-shaped and swollen cells followed by cell lysis (Fig. 4D and 5A and data not shown). Analysis of electron micrographs revealed the formation of cell membrane-associated vacuoles (20% of cells; n = 124) which were not seen in comparable electron micrographs of wild-type spores outgrowing in PAB medium with 500 mM NaCl and 10 mM MgCl₂ (Fig. 5A and data not shown). When outgrowing spores lacking PBP1 or PBP1 and either PBP2c, -3, or -4 were germinated in PAB medium with 500 mM NaCl, very little elongation took place and the OD₆₀₀ did not increase substantially (Fig. 3B, 4E, and 5B and data not shown). The inclusion of 10 mM Mg²⁺ in PAB medium with 500 mM NaCl prevented the lysis of outgrowing wild-type spores and allowed normal outgrowth of ponA spores (Fig. 3C). Examination of cultures of outgrowing wild-type and ponA spores 150 min after the initiation of spore germination in the latter medium showed that rod-shaped cells were present (>98%) (Fig. 4F and data not shown). Outgrowth of wild-type spores in PAB medium with 1.0 M sorbitol did not result in the drop in OD₆₀₀ observed during outgrowth in PAB medium with 500 mM NaCl, and elongation of ponA spores did take place (data not shown). Microscopic examination of wild-type cultures 180 min after the initiation of spore germination in PAB with 1.0 M sorbitol revealed a cell morphology similar to that of wild-type spores undergoing outgrowth in PAB alone (data not shown). However, the percentage of bent forms in cultures of ponA spores after 180 min of outgrowth in PAB medium with 1.0 M sorbitol (44%; n = 217) was increased somewhat over that of ponA spores undergoing outgrowth in PAB medium alone (27%; n = 207), although the percentages of filamentous cells were similar (13 and 14%; n = 217 and 207, respectively).

FIG. 5.

Electron microscopy of wild-type (A) and ponA (B) spores during outgrowth in PAB medium plus 500 mM NaCl. Spore germination and outgrowth were accomplished as described in Materials and Methods. Spores were harvested by centrifugation 130 min after the initiation of spore germination, fixed, and prepared for electron microscopy as described in Materials and Methods. The vacuoles associated with the walls of some cells (20%; n = 124) in panel A are indicated by arrows. The scale is the same for both panels. Bars, 1 μm.

When ponA spores were germinated in 2×YT medium, which has more Mg²⁺ and Ca²⁺ than PAB medium, fewer bent cells (5.4%; n = 251) were generated than in PAB medium. Spores lacking PBP1 and PBP4 also elongated more efficiently in 2×YT medium but generated a significant population (20%; n = 269) of tightly coiled helical cells 150 min after the initiation of spore germination (Fig. 4G). An additional population of bent cells that did not form tight helices was present (31%; n = 269) (data not shown). The addition of 10 mM Mg²⁺ to 2×YT medium prevented the formation of both helical and bent cells from spores lacking PBP1 and PBP4 (data not shown).

DISCUSSION

It has been found previously that disruption of the single ponA homolog in N. gonorrhoeae species is lethal, as is disruption of both ponA and ponB in E. coli (19, 41, 53). While B. subtilis cells with a deletion of the ponA gene, encoding PBP1, will grow in rich medium (34), this work demonstrates that deletion of ponA prevents vegetative growth of B. subtilis unless appropriate amounts of either Mg²⁺ or Ca²⁺ are present. While we do not know the exact mechanism by which divalent cations promote growth of ponA cells and suppress morphological defects in both wild-type and ponA strains, we present several possibilities. First, a threshhold level of Mg²⁺ may be required for cell division to occur in B. subtilis during both vegetative growth and spore outgrowth. It has been reported that Bacillus species grown in media with low levels of Mg²⁺ produce filaments (51), suggesting a requirement for Mg²⁺ in septum formation. In addition, the decreased growth rate and spherical-cell formation of B. subtilis rodB mutants (rodB is allelic to mreD [49]) are suppressed by increased [Mg²⁺], although the presence of specific anions is also required (39). Interestingly, MreD is predicted to be a transmembrane protein possibly involved in cell division (49). In E. coli, PBP1A and PBP1B have been hypothesized to play a role in the initiation of septation (9, 10); the filamentation seen in B. subtilis ponA mutants at low [Mg²⁺] suggests that a similar role is possible for B. subtilis PBP1. However, any involvement of PBP1 in septation can clearly be compensated for in its absence, since septa are formed appropriately at higher Mg²⁺ concentrations. A second possibility is that a threshhold level of Mg²⁺ is required for proper PG synthesis and this threshhold is increased in the absence of PBP1. Indeed, it has been reported that B. subtilis cells grown in Mg²⁺-deficient medium accumulate PG precursors (11).

The helical morphology observed during outgrowth of spores lacking PBP1 and PBP4 in 2×YT medium confirms a previous report of a redundancy in function for these two proteins (38). Interestingly, this helical-cell phenotype has been previously reported in wild-type B. subtilis cells grown in a chemostat in a medium with a low [Mg²⁺] (43); B. subtilis cells treated with penicillin G or chlorpromazine (which interacts with cell membranes) (47); mutants resistant to Triton X-100 (47) (interestingly, some of the latter strains were grown in PAB medium); strains with a conditional mutation in pbpB, encoding PBP2b (44); and a strain with mutations in prfA, ponA, pbpD, and pbpF (38). However, with the exception of the chemostat experiments, it has not been determined whether the helical-cell phenotype is suppressed by Mg²⁺.

One model for gram-positive cell wall formation suggests the rotation of one cell pole around the other during PG synthesis (20). This rotation might form helical cracks on the outer PG wall, generating a stressed PG which is susceptible to autolysin activity. The appropriate balance of autolysin and cell wall-synthetic activities would lead to straight-rod formation, while an alteration in either of these activities might result in the formation of helical or bent cells (20). It cannot be ruled out a priori that the reason that low levels of divalent cations cause a bent- or helical-cell morphology is that some alteration in autolysin activity occurs. However, since several mutations previously found to result in helical cells alter enzymes involved in PG synthesis, as do mutations in the present work, we speculate that the alteration in cell morphology in ponA mutants caused by growth in media with low [Mg²⁺] may involve a perturbation of PG synthesis, perhaps through changes in the properties of the cell membrane. Divalent cations may suppress these morphological phenotypes by (i) changing membrane properties, which in turn alters cell wall biosynthetic enzymes; (ii) stabilizing a membrane-bound PG-synthetic enzyme complex (which may be altered by the absence of PBP1); (iii) allowing the proper orientation of a PG-synthetic enzyme complex within the cell membrane; (iv) facilitating interaction between PG-synthetic enzymes and their substrates; or (v) being directly required for cell wall-biosynthetic activity. Additionally, it appears that the mechanism by which Mg²⁺ acts to facilitate both cell growth and rod shape is antagonized by high concentrations of salt, even in wild-type cells. Indeed, autolysis of B. subtilis cells in the presence of 100 mM monovalent cations was previously reported; this autolysis was suppressed by the addition of 100 mM divalent cations, carbon, or nitrogen (46). It has also been reported that high levels of NaCl can compete for Mg²⁺ that is normally adsorbed to the cell walls in B. subtilis (25). Consequently, high NaCl levels may effectively increase a cell’s requirement for divalent cations and activate autolysins in cells growing in an environment with a low level of divalent cations (46).

The possibility that differences in three-dimensional PG structure alter divalent-cation binding such that elevated levels of Mg²⁺ and Ca²⁺ are required to stabilize PG formed in ponA mutants also cannot be ruled out, since Mg²⁺ and Ca²⁺ bind PG of both E. coli and B. subtilis (3, 16). Divalent cations also have a high affinity for the anionic polymers in teichoic acid (14), and we cannot exclude the possibility of an alteration in this interaction in the ponA strain. However, we do not favor this hypothesis because when Mg²⁺ is added to helical cells of strain PS2182 lacking PBP1 and PBP4, suppression of the helical-cell phenotype is not seen until after at least 30 min (data not shown). Although further work is required to characterize the exact role of Mg²⁺ and Ca²⁺ in promoting cell growth and normal morphology in B. subtilis, clearly it would be interesting to examine whether high levels of divalent cations are also able to suppress the lethal effect of the loss of all PBP1 in other bacteria.