The Cell Wall Regulator σ^I Specifically Suppresses the Lethal Phenotype of mbl Mutants in Bacillus subtilis

Bacterial strains, plasmids, and oligonucleotides.The B. subtilis strains and plasmids used in the present study are listed in Table 1, and the oligonucleotides are listed in Table 2.

General methods.Liquid cultures for B. subtilis strains were grown in Difco Antibiotic Medium 3 (PAB) at 37 or 50°C as indicated. Nutrient agar (NA; Oxoid) plates were used for growth on solid medium. Minimal concentrations of Mg²⁺ required for growth were determined on NA or modified salts medium (7). MgSO₄ was added as a source of Mg²⁺. DNA manipulations and Escherichia coli DH5α transformations were carried out using standard methods (50). B. subtilis strains were transformed according to the method of Anagnostopoulos and Spizizen (2) as modified by Jenkinson (32). Selection for B. subtilis transformants was carried out on NA, supplemented with antibiotics, as required: kanamycin (5 mg ml⁻¹), chloramphenicol (5 mg ml⁻¹), erythromycin (1 mg ml⁻¹), lincomycin (25 mg ml⁻¹), and/or spectinomycin (50 mg ml⁻¹). IPTG (isopropyl-β-d-thiogalactopyranoside) at 1 mM was added as indicated.

Screen for Mg²⁺-independent suppressor mutants.Random transposon mutagenesis was performed using the mariner-based transposon tnYLB-1 as described previously (38). In short, the plasmid pMarB was introduced into an mbl mutant strain (2505) at 30°C in the presence of high Mg²⁺ concentrations. Individual colonies were picked, grown in LB medium at 37°C to induce transposition for 8 h, and then plated on NA plates not supplemented with Mg²⁺ but containing kanamycin to select for the transposon insertions creating Mg²⁺-independent strains. Individual colonies were picked, and deletion of mbl (Spc^r), integration of the transposon tnYLB-1 (Neo^r), and loss of the plasmid (Erm^s) were checked by patching on plates containing the appropriate antibiotic. Linkage between transposon insertion and Mg²⁺ independency was verified by back-crossing chromosomal DNA of single colonies three times into an mbl mutant background. Ten potent suppressors were chosen, and the site of transposon insertion was determined by inverse PCR amplification and sequencing using oligonucleotide primers IPCR1 to IPCR3 as described previously (38).

Construction of insertional deletion mutants.Chromosomal regions of 2.5-3 kb flanking the gene(s) to be deleted were PCR amplified using the primer pairs A/B and C/D (rsgI-A/rsgI-B and rsgI-C/rsgI-D for the rsgI deletion, sigI-A/sigI-B and rsgI-C/rsgI-D for the sigI rsgI double deletion, mreBH-A/mreBH-B and mreBH-C/mreBH-D for the mreBH deletion, mbl-A/mbl-B and mbl-C/mbl-D for the mbl deletion, and LENm1/LENm2 and LENm3/LENm4 [37] for the mreB deletion). These PCR products were then ligated to antibiotic resistance cassettes (neo from pBEST501 [31], erm from pMUTIN4 [56], and cat from pCotC [58]) and reamplified using the outside primer pair B/D (LENm1/LENm4, respectively). Transformation of the resulting PCR products into B. subtilis 168 or, in case of the mreB deletion, into strain 3728, with selection for the appropriate antibiotic then gave rise to strains where the target gene is substituted by an antibiotic resistance cassette. Deletion of the gene(s) and insertion of the resistance cassette were verified by PCR.

Construction of overexpression constructs and pLOSS*sigI.For overexpression constructs, sigI or rsgI were PCR amplified using the primer pairs sigI-fw/sigI-rev and rsgI-fw/rsgI-rev. The PCR products and the plasmid pPL82 (47) were digested with HindIII and SphI and ligated. The resulting plasmid with the sigI or rsgI under the control of the inducible P_spacHY promoter was then transformed into B. subtilis, selecting for resistance to chloramphenicol.

To test for synthetic lethality, we constructed pLOSS* carrying sigI (pSG5916). Therefore, sigI was amplified with its native promoter using the primers sigI-prom and sigI-rev. The PCR product and pLOSS* were digested with NheI and SphI, ligated, and the resulting plasmid was transformed into the desired B. subtilis strains selecting for spectinomycin resistance. The addition of spectinomycin is required to maintain the unstable plasmid in most strain backgrounds.

Phenotypic characterization.Sensitivity against cold stress was tested by comparing the number of CFU after incubation overnight at 37°C and after 3 days at 4°C as described previously (28). The width of membrane stained cells was measured by using ImageJ (http://rsb.info.nih.gov/ij/ ).

Microscopic imaging.For microscopy, cells from an overnight liquid or solid culture were diluted into PAB medium, supplemented with 20 mM MgSO₄ when required, and grown at 37 or 50°C. Cells were mounted on microscope slides covered with a thin film of 1% agarose in minimal medium (23). Staining of the membrane was achieved by adding 2 μl of Nile red (Molecular Probe) solution (12.5 mg ml⁻¹) to the agarose slide. Nucleoids were stained by mixing 8 μl of the cell suspension with 2 μl of DAPI (Sigma) solution (1 mg ml⁻¹ in 50% glycerol) before mounting the sample on the agarose covered slide. Images were acquired with a 14 Sony CoolSnap HQ cooled charge-coupled device camera (Roper Scientific) camera attached to a Zeiss Axiovert M135 microscope or a Zeiss 15 Axiovert 200M microscope. ImageJ was used to analyze the images, and manipulation was limited to altering brightness and contrast to obtain optimal prints.

Lethal effects of mbl deletion can be rescued by high concentrations of magnesium.The actin homologue Mbl has been described as being nonessential in B. subtilis (1, 33), but the authors of those studies had already indicated that mbl mutants are slow growing and tend to pick up mutations that enhance growth. The reported Mg²⁺ dependency of both mreB (20) and mreBH (though only at low levels) (7) mutants led us to reconstruct the mbl deletion strain in the presence of 20 mM Mg²⁺. Selecting for transformants under these conditions resulted in a 10-fold increase in plating efficiency, giving relatively small but uniformly shaped colonies (Fig. 1A). The strain grew well on NA plates supplemented with Mg²⁺ but failed to grow on unsupplemented plates (Fig. 2A). Also, in liquid culture (PAB medium) an elevated Mg²⁺ concentration greatly improved the growth rate (Fig. 1B). Microscopic examination of mutant and wild-type cells revealed the characteristic twisted and bloated morphology of the mutant in unsupplemented medium (Fig. 1C); however, in the presence of Mg²⁺ the cell morphology was greatly improved (Fig. 1D). Nevertheless, under high Mg²⁺ conditions the mbl mutant cells still differed from the wild-type (Fig. 1E and F) in two ways: first, they were slightly bent and irregularly shaped, and second, their average diameter was ca. 12% greater (Table 3) . Wild-type cells had their typical straight rod morphology under both conditions (Fig. 1E and F).

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FIG. 1.

B. subtilis Δmbl is Mg²⁺ dependent. (A) Plating efficiency after transformation selecting for deletion of mbl with (left) or without (right) addition of 20 mM Mg²⁺. (B) Growth curve of B. subtilis wild type (open symbols) and mbl mutant (closed symbols) at 37°C in PAB medium without (circles) or with (triangles) addition of 20 mM Mg²⁺. (C to F) Morphology (phase-contrast microscopy) of B. subtilis Δmbl grown in PAB (C) or in PAB supplemented with 20 mM Mg²⁺ (D) compared to a wild-type strain grown in PAB (E) or in PAB containing 20 mM Mg²⁺ (F). Scale bar, 5 μm.

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FIG. 2.

Heat stress (50°C) rescues the mbl mutant. (A) Growth of B. subtilis wild type and the Δmbl, ΔrsgI, and Δmbl ΔrsgI mutants on NA plates after incubation at 37°C (top left), 50°C (top right), and NA supplemented with 20 mM Mg²⁺ at 37°C (bottom). (B) Growth curves of B. subtilis wild-type (⧫), mbl mutant (▴), and mreB mutant (▪) in PAB medium incubated at 50°C. (C and D) Images of B. subtilis wild-type (C) and mbl mutant (D) grown in PAB medium at 50°C. Scale bar, 0.5 μm.

A screen for Mg²⁺-independent suppressor mutants of B. subtilis Δmbl. To gain insights into the function of Mbl, we screened for mutants in which the Mg²⁺ dependency of the mutant was suppressed. The plasmid pMarB carrying the mariner transposon (37) was introduced into a freshly constructed Δmbl strain background in the presence of 20 mM Mg²⁺. A library of approximately 60,000 mutants was plated with selection for Mg²⁺-independent growth. Ten strains with potent suppressor mutations were chosen and checked for linkage of the transposon insertion to the suppression phenotype by three consecutive back-crosses into the Δmbl mutant background. The sites of transposon insertion were determined by sequencing the products of inverse PCRs using primers IPCR1 to IPCR3 (37).

All of the insertions found appeared to severely truncate their respective open reading frames and thus are likely to represent null alleles of the genes. In two of the ten selected suppressor strains, the transposon was found to have inserted independently into the rsgI gene (previously ykrI), encoding the anti-sigma factor for σ^I (3). Three independent insertions were in yflE, encoding a homologue of the lipoteichoic acid synthase LtaS from S. aureus (24; K. Schirner et al., unpublished data). Two independent insertions were found in ylxA (synonyms yllC or mraW), which lies in an operon with yllB, ftsL, and pbpB and encodes a protein of unknown function. However, the ylxA deletion proved to be not very potent in suppressing the Mg²⁺ dependency of the mbl deletion strain (data not shown). One transposon insertion each was found in yaaT encoding a protein involved in the phosphorelay cascade during initiation of sporulation (29), in the gene for the glutamate transporter GltT (51, 54), and in pnpA which codes for polynucleotide phosphorylase (39, 43, 59). We focus in the present study on the most potent suppressor, generated by insertion in rsgI.

Increased activity of σ^I rescues the B. subtilis mbl mutant.The rsgI gene consists of 1,143 bp, encoding a protein of 381 amino acids. In two independent strains the transposon had inserted in rsgI after bp 2 and 518, respectively, suggesting that suppression worked by eliminating the function of rsgI. Sequence analysis using the published genome sequence of B. subtilis on the SubtiList webserver revealed that rsgI overlaps by 1 bp the sigI gene, coding for the alternative sigma factor σ^I, and the two genes have been shown to be cotranscribed (3). Because sigI is upstream of rsgI, its transcription is unlikely to be impaired by insertion of the transposon in the open reading frame of rsgI.

An rsgI-null mutant was constructed (strain 4263) and the mutation was introduced into an mbl deletion strain, generating strain 4266. Growth of this strain on NA confirmed that deletion of rsgI suppresses the Mg²⁺ dependency of the mbl mutant (Fig. 2A).

Light microscopy showed that, compared to the mbl single mutant, lysis occurred only rarely, and cell width was restored to the wild-type level (Table 3). Nevertheless, the double-mutant cells still exhibited a degree of twisting (Fig. 3D).

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FIG. 3.

Deletion of rsgI suppresses the Mg²⁺ dependency of B. subtilis Δmbl. (A to D) Phase-contrast microscopic images of B. subtilis wild-type (A), rsgI mutant (B), sigI rsgI double mutant (C), and mbl rsgI double mutant (D) grown in PAB at 37°C. Scale bar, 5 μm. (E) Growth curves of B. subtilis wild-type (⧫), ΔrsgI (▪), ΔsigI rsgI (•), Δmbl (▴), and Δmbl ΔrsgI (*) in PAB at 37°C. (F) Survival of wild type, sigI rsgI mutant, and rsgI mutant after cold stress (3 days at 4°C) indicated as a percentage.

Since RsgI has recently been shown to be an inhibitor of σ^I (3), it seemed likely that suppression was due to upregulation of σ^I activity. We constructed a strain overexpressing sigI by placing an additional copy of the gene under the control of the P_{spac HY} (47) promoter at the amyE locus. As anticipated, overexpression of σ^I also suppressed the Mg²⁺ dependency of B. subtilis Δmbl (data not shown). Even without the addition of inducer (IPTG) this strain (i.e., strain 4269) was viable on NA plates, probably because the promoter is poorly repressed and slight overexpression of σ^I is sufficient to rescue the mbl mutant.

Transcription of the sigI gene is induced by high temperature, and its gene product is a σ⁷⁰-type sigma factor involved in the heat shock response (3, 64). Because upregulation of σ^I expression, either by deletion of the anti-sigma factor RsgI or by overexpression of sigI, was able to suppress the Mg²⁺ dependence of the mbl mutant, we predicted that upregulation of σ^I by heat stress should also suppress the Δmbl phenotype. Indeed, when incubated at 50°C we found that the mbl mutant no longer requires Mg²⁺ to form colonies on NA plates (Fig. 2A). Also, the growth rate in PAB medium at 50°C was indistinguishable from that of the wild-type (Fig. 2B). Under these conditions both wild-type (Fig. 2C) and mbl mutant (Fig. 2D) strains grew in chains and showed a degree of twisting around their long axis. However, the mbl mutant still had a significantly greater (ca. 115%) cell width (Table 3).

Expression of the σ^I regulon has been shown to be strongly repressed in the presence of 0.5% glucose (3). However, when grown at high temperature the Mg²⁺ dependency of the B. subtilis Δmbl strain was still suppressed on NA plates containing 0.5 or 1% glucose. Perhaps the repressed level of σ^I expression is sufficient to rescue the mbl mutant. Alternatively, other stress-sensing systems, such as other alternative sigma factors or two-component systems, might have overlapping functions with the σ^I regulon enabling mbl mutant growth even under conditions of σ^I repression (34, 40).

It has previously been shown that mreB deletion strains were only viable on plates containing high Mg²⁺ concentrations (20). Therefore, we tested whether activation of σ^I also enabled Mg²⁺-independent growth of this strain by constructing a double deletion ΔmreB ΔrsgI strain (strain 4267). This strain was able to grow on unsupplemented NA plates; in liquid cultures, however, no suppression was observed (Table 4). Inducing σ^I activity by incubation at 50°C did not restore growth of the mreB deletion strain either on plates or in liquid culture (Fig. 2B and data not shown). A strain with a deletion of mreBH does not show a clear phenotype under the growth conditions described, and the double deletion of both mreBH and rsgI (4268) had no phenotypic effect (Table 4). We therefore conclude that the rsgI deletion is a specific suppressor of mbl.

Phenotypic characterization of sigI upregulation and deletion.If σ^I activation suppresses the mbl phenotype by making a compensatory change in the cell wall structure, this change might be visible in rsgI single mutants. Therefore, we characterized the phenotypes of strains with an rsgI single deletion and with a sigI rsgI double deletion.

Neither sigI nor rsgI is essential under normal growth conditions or when incubated at 50°C (3, 64). The growth rates of B. subtilis 168 and the ΔrsgI and ΔsigI rsgI strains in PAB medium at 37°C were indistinguishable (Fig. 3E). By light microscopy both mutants showed normal cell morphology (Fig. 3B and C), However, the cell diameter of strain 4263 (ΔrsgI) was decreased by 12% compared to the wild type (Table 3). In contrast, the width of the sigI rsgI double mutant cells was not significantly different from that of the wild type.

Because σ^I activity enables cells to survive at high temperatures, we sought to determine whether changes induced by σ^I upregulation also affected the response to cold stress. Cold resistance of the strains was assayed by plating dilutions of liquid cultures and comparing a viable count with the colonies appearing after incubation at 4°C for 3 days (28). The survival rate of a wild-type strain was ca. 27%. Interestingly, the survival rate of the sigI rsgI double deletion strain was decreased to ca. 21%, while the rsgI mutant showed a twofold higher degree of cold resistance (53%) (Fig. 2F), indicating a role for σ^I in survival at low, as well as high, temperatures.

Recently, mreBH and bcrC (encoding an undecaprenyl pyrophosphate phosphatase [4]) have been shown to be members of the σ^I regulon (55) genes; both code for proteins connected to cell wall metabolism. Therefore, the authors of these studies suggested a role for σ^I in maintaining the cell envelope integrity during heat stress. Transmission electron microscopic images, however, failed to reveal differences in cell wall architecture between a wild-type strain and an rsgI mutant (data not shown).

Synthetic lethality of sigI mbl double mutants.Deletion of rsgI (resulting in σ^I activation) or overexpression of σ^I both resulted in the suppression of the Mg²⁺-dependent phenotype of an mbl deletion strain. In contrast, overexpression of the anti-sigma factor RsgI (via a P_spacHY rsgI construct, strain 4271) had a deleterious effect on mbl mutant cells even in the absence of inducer and presence of Mg²⁺ (data not shown). When mbl mutant cells were transformed with DNA from a strain carrying an overexpression construct P_spacHY rsgI, transformants only appeared after long incubation and were very small. Similar observations were made when the rsgI overexpression construct was introduced in an mreB deletion strain. In contrast, the same transformation into the wild type or a parent strain with a deletion of mreBH readily gave colonies. Consistent with this result, we were unable to construct a strain in which insertional deletions of sigI and mbl were combined unless an extra copy of sigI was placed at an ectopic locus (amyE::P_spacHY sigI, strain 4270).

Our lab has recently developed a system for screening for combinations of synthetically lethal gene deletions (9). To further investigate the putative sigI/mbl synthetic lethality, we constructed a derivative of the unstable plasmid pLOSS* (9) carrying the sigI gene controlled by its native promoter (pSG5916). pLOSS* also carries a lacZ gene under the control of a constitutive promoter (P_divIVA). Thus, colonies are blue on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) when the plasmid is retained; loss of pSG5916 is indicated by a white colony color. In a sigI rsgI double mutant background, pLOSS*sigI was readily lost, yielding white colonies, indicating that sigI is not essential under these conditions (Fig. 4A). In contrast, in cells additionally carrying a deletion of mbl (ΔsigI rsgI Δmbl) the plasmid was retained even in the presence of Mg²⁺ (large blue colonies with only occasional small white ones; Fig. 4B), while the mbl single mutant again readily lost the plasmid (white colonies) in the presence of Mg²⁺ (Fig. 4C shows that blue colonies were always formed when direct selection for the plasmid was maintained). Interestingly, the extra copy of sigI on the plasmid promoted the growth of an mbl single mutant giving small blue colonies on NA plates not supplemented with Mg²⁺. This is consistent with an upregulation of σ^I suppressing the phenotype of mbl mutant cells, because this strain carries two copies of the sigI gene. Similar to the mbl deletion background, pLOSS*sigI was retained in an ΔmreB ΔsigI rsgI background (data not shown), indicating the synthetic lethality of this combination of mutations.

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FIG. 4.

mbl and sigI are synthetically lethal. Strains with the unstable plasmid pSG5916 plated on NA plates containing X-Gal without (left) or with addition of 20 mM Mg²⁺ (middle) or containing both 20 mM Mg²⁺ and antibiotic (spectinomycin) selecting for the resistance cassette contained on pLOSS* and magnesium (right). A white colony color indicates that the cells have lost the unstable plasmid; a blue colony color shows maintenance of pSG5916.

We conclude that, although sigI is not essential under normal growth conditions (3, 64), at least part of the σ^I regulon is essential for viability of B. subtilis mbl and mreB deletion strains, even in the presence of high Mg²⁺ concentrations.

The mechanism by which Mg²⁺ enables the growth of the mbl mutant and other mutants is unknown. Nevertheless, we were able to show that Mg²⁺ does not work by inducing σ^I activity. Using a fusion of the sigI promoter region to the reporter gene lacZ, it was evident that both in wild-type (strain KA) and mbl deletion (strain 4278) background strains the expression level of sigI is similar when grown with or without the addition of 20 mM Mg²⁺ to the medium. Only deletion of rsgI (strain 4279) led to an increased expression of sigI (Fig. 5).

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FIG. 5.

Magnesium does not induce σ^I activity. Expression levels of sigI in wild-type (⧫), mbl mutant (▴), and rsgI mutant (•) backgrounds grown with (open symbols) or without (closed symbols) addition of 20 mM Mg²⁺ to the medium. All strains carried the sigI promoter region fused to the reporter gene lacZ at the amyE locus. (A) Growth curve of the strains; (B) β-galactosidase activity.

Viability of an mreB mbl mreBH triple-deletion strain conferred by upregulation of σ^I.Although the single mutants of each of the B. subtilis mreB homologues can all be rescued by addition of Mg²⁺, the only viable double mutant combination was mbl and mreBH (A. Formstone, unpublished data). Thus, any combination, including the mreB deletion, appeared to be lethal. This leads to the idea that the three proteins have partially redundant functions (14, 33). The ability of σ^I activation to rescue the mbl mutant led us to investigate whether this was also the case in the absence of MreB. Transformation of the suppressed mbl mutant (Δmbl ΔrsgI, strain 4266) with chromosomal DNA from strain 3728 carrying an in-frame deletion of mreB (20) resulted in the ready selection of transformants on plates supplemented with Mg²⁺ (strain 4274). Furthermore, a mutant with all three mreB homologues (mreB, mbl, and mreBH) inactivated was readily constructed by introducing the mreB deletion into strain 4276 (ΔmreBH Δmbl ΔrsgI). Consistent with observations made for depletions of MreB in both mbl and mreBH mutant backgrounds (Formstone, unpublished) the resultant strain (i.e., strain 4277) was strictly Mg²⁺ dependent and displayed a spherical cell morphology when grown on NA or in PAB medium at 37°C (Fig. 6). Similar phenotypes had been observed for strains carrying deletions of mreC or mreD (37). However, in contrast to these strains, the ΔmreB Δmbl ΔmreBH ΔrsgI mutant did not require the osmoprotectant sucrose.

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FIG. 6.

A strain lacking all three actin homologues is viable when σ^I is upregulated. Microscopic images of strain 4277 (Δmbl ΔmreB ΔmreBH ΔrsgI) by phase contrast, membrane stain (FM5-95), and DNA stain with DAPI. Scale bar, 0.5 μm.

In various organisms MreB has been suggested to have a role in chromosome segregation (12, 22, 36), although interpretation of these observations is complicated by the severe shape defects or by the presence of other paralogues. In B. subtilis, the reported results have been contradictory, and the experiments are complicated by the presence of three MreB-like proteins with partially overlapping functions. To observe nucleoid localization in the absence of all three mreB-like proteins, we stained cells of strain 4277 with DAPI and examined cells by phase-contrast and fluorescence microscopy. Although rigorous quantitation is difficult because of the cells’ tendency to cluster and clump, thorough examination of many fields of cells failed to reveal a significant number of anucleate cells (Fig. 6). Often multiple nucleoids were discernible, but this is probably due to the large size of the spherical cells (57, 63). These observations suggest that this strain lacking all three actin homologues is still able to grow and undergo cell division in such a way that all daughter cells receive genetic material.

Overlapping but distinct function of the actin homologues in B. subtilis.The finding that mutants of B. subtilis actin homologues MreB and MreBH are sensitive to a low Mg²⁺ concentration (7, 20) led us to reconstruct the mbl mutant in the presence of high concentrations of Mg²⁺. The increase in plating efficiency, uniformity of colony shape, and amelioration of the cell morphology recapitulated the earlier findings made for the mreB and mreBH mutants. However, the mutants vary in optimal levels of Mg²⁺: the mreBH mutant requires only about 100 to 200 μM Mg²⁺ for viability, and the cells display a reduced cell width (7); the mreB mutant has a higher requirement for Mg²⁺ (2.5 mM), and depletion of the cation results in an increase in cell diameter and ultimately lysis (20). Finally, the newly constructed mbl mutant requires the addition of about 3 mM Mg²⁺, which is in a range similar to that for the previously described mreB mutant. In unsupplemented medium the strain grows slowly, and the cells tend to twist, form chains, and swell over their length and are prone to lysis.

In an otherwise wild-type background, the only viable double mutant was the Δmbl ΔmreBH mutant, which has a phenotype similar to that of an mbl single mutant. Combinations with ΔmreB were lethal, and depletion of MreB in either mbl or mreBH mutant backgrounds led to a loss of rod shape and cell death (14; Formstone, unpublished) irrespective of Mg²⁺ levels. Thus, the three MreB-like proteins appear to have overlapping functions in cell elongation, because mreB is essential in strains deleted for any of the other two homologues. However, although the three mutants share certain characteristics such as the Mg²⁺ dependency and effects on cell shape, the phenotypic differences between the single mutants show that each has a partially differentiated function.

Mbl is dispensable under conditions of increased σ^I activity.We found two independent transposon insertions in rsgI (ykrI), although the screen for suppressor mutations of the Δmbl lethal phenotype was probably not saturated. This gene encodes the anti-sigma factor of the alternative sigma factor σ^I, and the absence of RsgI causes activation of σ^I (3). Because rsgI deletion proved to be a potent suppressor, we further characterized the effect of increased and decreased σ^I activity on the cells. The lower Mg²⁺ dependence of the mbl mutant upon induction of the σ^I regulon might be due to modifications of cell wall structure. This is consistent with the recent finding that bcrC and mreBH, which both are directly or indirectly (via LytE [7]) involved in the process of cell wall turnover, are members of the σ^I regulon (55).

Many sigma factors, especially of the extracytoplasmic function-type group, have been shown to regulate components of the cell wall synthetic machinery in response to various kinds of stress (17, 25, 27, 34, 40, 49), and regulatory overlap between them and other stress-sensing systems (such as two-component systems) is common (5). Thus, transcription of the target genes responsible for rescuing mbl mutants by σ^I upregulation might also be controlled by other stress-sensing systems, which might be an explanation for the mbl mutant growing at 50°C even if σ^I activity is repressed by glucose (3).

Our results show that in situations in which σ^I activity is increased, the presence of Mbl is not essential for the cell. On the other hand, a basal activity of σ^I is required in mbl or mreB mutants, implying that at least one member of the regulon is essential in these strain backgrounds, even in the presence of Mg²⁺. Identification of which gene(s) in the σ^I regulon are responsible for the suppression of the mbl deletion strain is an interesting question for future research. Screening for genes synthetically lethal with mbl and rsgI promises to be a useful approach to identify important target genes of the σ^I regulon. However, this approach would only give answers if a single gene was responsible for the observed effect.

A strain without actin homologues loses its rod shape but is still able to propagate.MreB-like proteins have been proposed to be involved in chromosome segregation at least in Caulobacter crescentus and E. coli (6, 22, 36). On the other hand, deletion of mreB did not impair chromosome segregation in Streptomyces coelicolor or Anabaena sp. (30, 41). The situation in B. subtilis has been controversial thus far: the discovery that the single mutants can be rescued by Mg²⁺ seemed to exclude a critical function in chromosome segregation, but potentially overlapping functions of the other homologues might mask the effect. Activation of σ^I by deletion of rsgI enabled us finally to construct a triple-null strain. The spherical cell shape of the mutant is reminiscent of the phenotypes reported for mreC, mreD, and rodA deletions (37; M. Leaver, unpublished data). However, in contrast to the proteins encoded by these genes, MreB-like proteins are cytosolic and unlikely to be directly involved in cell wall synthetic processes on the outside of the cytoplasmic membrane. This supports a likely primary role for these proteins in positioning or otherwise organizing the cell wall synthetic machinery. However, initial experiments to localize TagH-green fluorescent protein, which forms bands consistent with an underlying helical pattern in a wild-type background (19), or staining of the nascent peptidoglycan with fluorescent vancomycin (11) in the strain lacking actin homologues did not reveal any striking differences compared to other spherical mutants such as the ΔrodA mutant (M. Leaver, unpublished data) or the ΔpbpA ΔpbpH double mutant (60; Leaver, unpublished). These observations indicate that localization of cell wall synthetic proteins is disturbed in cells that have lost their rod shape, both in the presence and in the absence of actin homologues. Importantly, no increased number of anucleate cells was found, suggesting that B. subtilis can still divide and efficiently generate progeny with nucleoids even in the absence of MreB-like proteins.