Elimination of PBP1 suppresses the lethality of an mreB null mutation
It has been reported that the growth of mreB null mutants is restored in the presence of high concentrations of Mg2+ (Formstone and Errington, 2005). To gain insights into the molecular basis for the growth impairment and morphological defect caused by inactivation of MreB, we isolated extragenic transposon (TnYLB-1) insertions that restored viability of an mreB mutant plated under non-permissive conditions (without added Mg2+ or inducer). Strain YK400 (ΔmreB amyE::Pxyl-gfp-mreB) has a functional copy of mreB fused to gfp and controlled by the xylose inducible promoter, Pxyl. Strain YK400 was transformed with the transposon plasmid and transformants were plated on penicillin assay broth (PAB, Difco Antibiotic Medium 3) agar in the absence of Mg2+ or inducer. After screening a library that generated about 30 000 colonies in the presence of xylose, we identified nine mutants capable of growth in the absence of Mg2+ or inducer and which had stable suppressor mutations linked to a transposon insertion. Mapping and sequencing of five of the mutants showed that the transposon had inserted into the ponA gene. ponA encodes PBP1, which is a high-molecular-weight bi-functional PBP proposed to catalyse both the transglycosylation and transpeptidation of PG precursors (Murray et al., 1998; Pedersen et al., 1999). The other transposon insertions were in ptsI (three hits), and ccpA, and they are not discussed further here.
To confirm the ability of ponA disruption to rescue viability of the mreB mutant, a null mutation of ponA (ponA::spc) was transformed into the mreB mutant. As shown in Fig. 1A, an in-frame deletion mutant of mreB (3725, ΔmreB) grew on solid PAB medium supplemented with 10 mM MgSO4 (c), but not on unsupplemented medium (d). In contrast, the double mutant YK401 (ΔmreB ponA::spc) grew on solid PAB medium without added MgSO4 (Fig. 1A d). Growth of mreB mutant cells in liquid PAB after removal of extra Mg2+ resulted in cell lysis (Fig. 1B, open diamonds). However, the mreB ponA double mutant cells grew in liquid medium in the absence of Mg2+ (open circles), although more slowly than wild-type cells, confirming that lethality of the mreB mutation was largely suppressed by disruption of ponA.
Figure 1. Lethal phenotype of cells deleted for mreB, and rescue by the disruption of ponA. A. Growth on PAB agar plates supplemented with (a and c) or without 10 mM Mg2+ (b and d) of strains wild-type (168), ΔponA (4223), ΔponAΔpbpD (YK887), ΔponAΔpbpDΔpbpF (YK905), ΔmreB (3725), ΔmreBΔponA (YK401), ΔmreBΔponAΔpbpD (YK885) and ΔmreBΔponAΔpbpDΔpbpF (YK888). B. Growth curves in PAB liquid medium supplemented with (solid symbols) or without 10 mM Mg2+ (open symbols) of strains wild-type (squares; 168), ΔponA (triangles; 4223), ΔmreB (diamonds; 3725) and ΔmreBΔponA (circles; YK401).
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Mutual suppression of mutations affecting MreB and the bifunctional PBPs
To test whether the effects described above were specific for ponA we examined the effects of mutations affecting the two other major vegetatively expressed genes encoding class A PBPs in B. subtilis, pbpD (PBP4) and pbpF (PBP2c) (Popham and Setlow, 1993; 1994). Various mutant combinations were constructed and first tested for viability on PAB plates supplemented with or without 10 mM Mg2+(Table 1 and Fig. 1A). A ponA pbpD double mutant, and a ponA, pbpD and pbpF triple mutant both showed a strong growth defect in the absence of Mg2+, and the triple mutant barely grew even in the presence of Mg2+ (Table 1 and Fig. 1A a and b). Under our conditions, the growth defect was more severe than previously reported (McPherson and Popham, 2003) (see Fig. S1 and Discussion). The severe growth defect was consistent with the bi-functional PBPs having partially redundant roles in the essential transglycosylation reaction of PG synthesis. Interestingly, suppression of the lethal mreB phenotype in the absence of Mg2+ was not observed for pbpD or pbpF mutations, nor by simultaneous disruption of pbpD and pbpF (Table 1); it occurred only when these mutations were combined with ponA (Table 1, Fig. 1A d and Fig. S1) (disruption of pbpA or pbpH, which encode class B PBPs, also did not suppress the lethality of mreB mutant; data not shown). These results suggested that PBP1 activity is involved in lethality of mreB mutations at low Mg2+.
Table 1. Viability of mreB mutant cells in the absence of bi-functional PBPs.
|Strain||Viability on PAB plate|
| ||10 mM Mg2+|| ||10 mM Mg2+|
Surprisingly, these experiments also revealed a strong mutual suppression effect, in that mutation of mreB strongly enhanced the growth of the ponA mutants and the derivatives in which other class A PBPs were knocked out. Note that the triple PBP mutant hardly grew at all on a Mg2+-supplemented plate when MreB was present, whereas the equivalent MreB- strain grew well even in the absence of Mg2+ (Fig. 1A b and d and Fig. S1). Therefore, the deleterious consequences of loss of the three major class A PBPs can be largely overcome by deletion of mreB.
Bulging and lysis of mreB mutants depends on PBP1 function
Previous experiments showed that mreB mutant cells undergo bulging and eventually lyse under normal Mg2+ conditions (Formstone and Errington, 2005). In addition, we recently reported that PBP1 activity is required for bulging of gpsB ezrA double mutants (Claessen et al., 2008). To test whether this was also the case for bulging of mreB mutants, various strains were examined by phase contrast microscopy of cultures grown in PAB medium supplemented with sucrose, which prevents lysis of the mreB mutant (Formstone and Errington, 2005). The mreB mutant cells maintained their rod shape reasonably well in the presence of 10 mM Mg2+ (Fig. 2B), whereas the cells became swollen after the removal of Mg2+, eventually undergoing a massive degree of bulging and lysis (Fig. 2C and D), as shown previously (Formstone and Errington, 2005). Ectopically expressed GFP-MreB (strain YK400, ΔmreB amyE::Pxyl-gfp-mreB) complemented the effects of the chromosomal mreB deletion preventing bulging and lysis (data not shown), confirming that the bulging phenotype is caused by inactivation of MreB. In contrast, in the absence of both PBP1 and MreB, no bulges were detected after the removal of Mg2+ (Fig. 2G and H), although many cells had an abnormal coiled morphology (Fig. 2H). As noted previously, cells of the ponA single mutant were slightly thinner and longer than wild-type cells (Murray et al., 1998; Claessen et al., 2008; compare Fig. 2A and E).
Figure 2. Morphological effects of the mreB ponA double mutant. Phase-contrast images of cells in wild-type (168; A, I–K), ΔmreB (3725; B–D and M–O) ΔponA (4223; E and L) and ΔmreBΔponA (YK401; F–H and P). A–H. Cells were grown to exponential phase at 37°C in PAB liquid medium supplemented with (B and F) or without 10 mM Mg2+ (A, C–E, G and H). Image of B. subtilis cells were captured at 60 min (C and G) and 120 min (A, D, E and H) after the removal of Mg2+ from PAB medium. I–P. Cells were grown to exponential phase at 37°C in minimal medium supplemented with 10 mM Mg2+ and they were diluted into the fresh minimal medium supplemented with 10 mM Mg2+ (I and M), 1 mM Mg2+ (J and N) and 0.1 mM Mg2+ (K, L, O and P). Images were captured at 90 min after dilution of the cells. Scale bars represent 5 μm.
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The morphology of mreB mutant cells was further examined in a synthetic minimal medium with various concentrations of Mg2+. In high-Mg2+ media (10 mM, Fig. 2M; or 5 mM, data not shown), the mreB mutant cells maintained their rod shape. However, at 1 mM the cells frequently showed polar bulges (Fig. 2N). Such bulges were not observed in cells of the wild-type strain in 1 mM (Fig. 2J) or 0.5 mM (see below) Mg2+ medium, but, interestingly, at lower concentrations of Mg2+ (0.1 mM, Fig. 2K; or 0.05 mM, Fig. 3C) polar bulges were observed, and the cells were not viable in 0.01 mM Mg2+ medium (data not shown). In contrast, no bulges were detected in ponA-disrupted cells cultivated under even lower-Mg2+ conditions (Fig. 2L and P). Thus, PBP1 is required for bulge formation in wild-type cells at low Mg2+ conditions, as well as in mreB-disrupted cells.
Figure 3. Localization of GFP–PBP1 in the mreB mutant cells. Localization of GFP–PBP1 in wild-type cells (YK706; A–C), mreB mutant cells (YK704; D–F and I) and gpsB ezrA double mutant cells (YK817; G and H). Cells were grown to mid-exponential phase at 37°C in minimal medium supplemented with 10 mM Mg2+ and they were diluted into the fresh minimal medium supplemented with 10 mM Mg2+ (A, D and G), 0.05 mM Mg2+ (B, C, E and F) and 0.5 mM Mg2+ (H and I). Fluorescence images were captured at 60 min (B and E) and 90 min (A, C, D, F and G–I) after dilution of the cells. Enlarged images are shown below (ii). Arrows indicate unusual polar localization of PBP1 in the wild-type cells under lower-Mg2+ conditions (B) or bulges pole in gpsB ezrA mutant cells (H). Scale bars represent 5 μm.
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Polar bulging is associated with abnormal localization of PBP1
It has been shown that PBP1 dynamically relocates from the septum to the lateral wall during the cell cycle and suggested that incorrect localization of PBP1 was responsible for the bulging phenotype of ezrA gpsB mutants (Claessen et al., 2008). Therefore, we suspected that bulge formation in mreB mutant cells or in the wild-type cells in low-Mg2+ medium might be due to delocalization of PBP1. Cells expressing GFP–PBP1 were cultivated in minimal medium with various concentrations of Mg2+. In mreB+ cells grown in medium supplemented with 10 mM MgSO4, GFP–PBP1 (YK706, amyE::Pxyl-gfp-ponA) was detected at the septum as a band, or in dots along the cylindrical parts of the cell as described previously (Claessen et al., 2008; Fig. 3A). At 60 min after reduction to 0.05 mM Mg2+, GFP–PBP1 remained predominantly in the cylindrical parts of the cell (Fig. 3B), although abnormal polar localizations were also detected (arrows). Further cultivation of mreB+ cells at this lower level of Mg2+ resulted in polar bulges and the discrete localizations of GFP–PBP1 along the lateral cell wall had virtually disappeared, being replaced by prominent fluorescence around the bulging poles (Fig. 3C). In mreB mutant cells, typical localization patterns of GFP–PBP1 were also observed in the presence of 10 mM Mg2+ (Fig. 3D). However, 60 min after reduction of the Mg2+ concentration, the cells already showed severe bulging and GFP–PBP1 fluorescence was predominantly at division sites and the bulging poles (Fig. 3E). By 90 min the cells were starting to lyse and GFP–PBP1 had largely condensed into single foci (Fig. 3F). Use of the fluorescent penicillin analogue Bocillin FL (Zhao et al., 1999) suggested that PBP1 is properly folded and inserted into the membrane in mreB mutant cells under low-Mg2+ conditions (Fig. S2). We conclude that PBP1-dependent polar bulging is associated with abnormal localization of PBP1 at the cell poles.
Lateral wall localization of PBP1 depends on MreB
Recent finding that simultaneous disruption of gpsB and ezrA results in abnormal polar localization of GFP–PBP1 suggests that GpsB and EzrA have some role in the localization dynamics of PBP1 during the cell cycle (Claessen et al., 2008). However, in that work, the localization of GFP–PBP1 in a gpsB and ezrA double mutant was only tested in medium with high Mg2+ (Claessen et al., 2008). Therefore, we examined the localization of PBP1 in gpsB and ezrA double mutant cells cultivated with lower concentrations of Mg2+. Cells of the mutant cultivated in minimal medium showed bulging poles by 90 min after reduction of the Mg2+ concentration (from 10 to 0.5 mM) (Fig. 3H and arrows), and accumulation of GFP–PBP1 fluorescence at the bulging poles was observed as shown previously (Claessen et al., 2008; Fig. 3H b). However, the discrete localizations of GFP–PBP1 along the lateral cell wall were still present in the cells (Fig. 3H). In contrast, in the mreB mutant cells, GFP–PBP1 fluorescence in the lateral wall was almost undetectable under the same conditions (Fig. 3I), suggesting that MreB is important for recruitment of PBP1 to the cylindrical parts of the cell.
PBP1 localization is independent of the MreB paralogues, Mbl and MreBH
Bacillus subtilis has three MreB isoforms, MreB, Mbl and MreBH. They have been demonstrated to colocalize in a single helical structure and to have important roles for cell shape determination (see Introduction). Therefore, we thought that Mbl and MreBH might also have a role in the regulation of PBP1 localization. To test this possibility, we examined cell morphology and localization of PBP1 in mbl and mreBH mutants in minimal media. In the presence of 0.5 mM Mg2+, mreB mutant cells showed polar bulges (Fig. 4B), but such bulges were not observed in the mbl and mreBH mutant cells (Fig. 4C and D). Under these conditions, discrete localizations of GFP–PBP1 on the lateral cell wall were clearly visible in mbl or mreBH mutants (Fig. 4E and F), but not in the mreB mutant cells (Fig. 3I). However, further reduction of the available Mg2+ (below 0.1 mM) resulted in a bulging phenotype and loss of lateral localizations of GFP–PBP1 in mbl or mreBH mutant cells (data not shown). Therefore, Mbl or MreH might be required for normal cell morphology and localization of PBP1 at very low Mg2+ concentrations (< 0.1 mM), while MreB is required at higher Mg2+ concentrations (< 1 mM: Fig. 2N).
Figure 4. No significant effect of the mbl and mreBH disruption on localization of PBP1. Cells of the wild-type (168; A), mreB mutant (3725; B), mbl mutant (4261 and YK811; C and E) and mreBH mutant (4262 and YK813; D and F) were grown to mid-exponential phase at 37°C in minimal medium supplemented with 10 mM Mg2+ and they were diluted into the fresh minimal medium supplemented with 0.5 mM Mg2+. Fluorescence images were captured at 90 min after dilution of the cells. A–D. The cell membranes of typical fields of cells are stained by Nile Red (A–D). E and F. GFP–PBP1 localization in the absence of Mbl and MreBH. Enlarged images are shown below (ii). Scale bars represent 5 μm.
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PBP1 associates with MreB in a complex
The above results showed that disruption of mreB under low Mg2+ causes delocalization of PBP1, bulging and finally cell death. Previous work suggested that PBP1 associated with the MreB helix of the lateral wall indirectly via an interaction with MreC (Claessen et al., 2008). To test for the existence of complexes containing both MreB and PBP1, we made an mreB-histidine tag construct, under the control of the xylose-inducible promoter, which was integrated into the amyE locus of ΔmreB strain (3738, ΔmreB amyE::Pxyl-mreB-his10). The growth rate and cell shape of 3738 were indistinguishable from that of the wild type in the presence of inducer, indicating that the fusion is functional (data not shown). Using this background, the only copy of ponA was modified such that it was under the control of the isopropyl β-D-thiogalactoside (IPTG)-inducible Pspac promoter (YK781, ΔmreB amyE::Pxyl-mreB-his10 Pspac-ponA). Cells expressing the MreB-His fusion were treated with the fluorescent penicillin analogue Bocillin FL (Zhao et al., 1999), and formaldehyde to cross-link protein, then disrupted by sonication. MreB complexes were purified using Ni resin under denaturing conditions, then heated to disrupt cross-linked proteins and separated by SDS-PAGE. As a negative control, cells expressing His-tagged DnaC [a component of the replisome that interacts with DnaI (Imai et al., 2000; Bruand et al., 2001; Ishikawa et al., 2006)] were analysed in parallel. As reported previously, DnaI was detected as a component of DnaC complex by liquid chromatography-tandem mass spectrometry analysis (Fig. 5A, lane 2). However, no DnaI was detected in equivalent samples from the MreB-His complexes (data not shown). In contrast, TufA, which has been detected as non-specific background in FtsA and DnaC complexes purified by similar methods (Ishikawa et al., 2006), was readily detectable in both complexes (Fig. 5A). Therefore, DnaI, at least, is specific to the DnaC complex. As an another control, the presence of MreC and SpoIIIE, which is DNA translocase and has N-terminal transmembrane segments that are required for proper septal localization (Wu and Errington, 1997; Sharp and Pogliano, 1999; 2002), in MreB complexes was analysed by Western blotting (Fig. 5B). MreC was readily detected in both the whole-cell extracts and in purified MreB complexes (Fig. 5B a), showing that MreB and MreC are physically associated in a complex. In contrast, SpoIIIE was not detected, or only present in trace amounts (Fig. 5B b), indicating that SpoIIIE is not closely associated with MreB.
Figure 5. Isolation and analysis of the MreB complex. A. Separation and visualization of protein complexes purified from cultures containing his-tagged MreB (strain YK781; lane 1) and DnaC (strain 168dnaCHis; lane 2) by SDS-PAGE and Colloidal Coomassie staining (see Experimental procedures). TufA (lanes 1 and 2), MreB-His (lane 1), DnaC-His (lane2) and DnaI (lane 2) were identified by mass spectrometry analyses (arrows). B. Whole-cell extracts (lanes 1) and purified MreB complexes (lanes 2 and 3) were separated, and proteins MreC (a) and SpoIIIE (b) were visualized by Western blotting using anti-MreC and anti-SpoIIIE antisera as described in Experimental Procedures. Samples amounting to 2.5 μg (lane 2) and 10 μg (lane 3) of total protein were loaded for purified MreB complexes. C. Detection of PBPs using the fluorescent penicillin analogue Bocillin FL. This analysis reveals several polypeptides covalently bound to Bocillin FL (Zhao et al., 1999). Cells of wild type (168; lanes 4 and 8), expressing MreB-His (YK781; lanes 1, 2, 5 and 6) and DnaC-His (168dnaCHis; lanes 3 and 7) were cultivated with (lanes 2–4 and 6–8) or without IPTG (lanes 1 and 5). Whole-cell extracts (lanes 1–4) and purified protein complexes (lanes 5–8) were separated and visualized as described in Experimental procedures. Proteins corresponding to the molecular masses of each PBP are indicated by arrows. Arrow heads indicate the position of copurified proteins with MreB-His. The asterisk shows a likely non-specific signal (see text). Several proteins were not detected in the MreB complex without PBP1 (open arrowheads), suggesting that they require PBP1 for the complex association with MreB or result from the degradation of PBP1.
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The scan for Bocillin fluorescence in the whole-cell extracts of B. subtilis strains revealed proteins corresponding to the molecular masses of PBP1, PBP2a, PBP2b, PBP2H, PBP2c, PBP4 and PBP5 (Fig. 5C, arrows). The most highly labelled and highest-molecular-weight band was not detected in cells expressing MreB-His cultivated in the absence of IPTG (i.e. with synthesis of PBP1 repressed) (Fig. 5C, lane 1), showing that this band corresponds to PBP1. When an MreB complex was purified in the presence of IPTG (i.e. in the presence of PBP1) using YK781, PBP1 was readily detected (Fig. 5C, lane 6). In contrast, PBP1 was not detected in the DnaC complexes (Fig. 5C, lane 7). Thus, we concluded that PBP1 is specific to the MreB complexes. In addition, several extra bands were also detected in the MreB complexes (Fig. 5C, arrow heads). Three of these were detected in complexes isolated from cultures with IPTG (Fig. 5C, open arrow heads), but not without IPTG (Fig. 5C, lane 5), suggesting that they are either degradation products of PBP1, or as yet unidentified PBPs that only associate with MreB when PBP1 is present. Three other fluorescently labelled bands corresponding approximately to the molecular masses of PBP2A, PBP2c and PBP4 were also detected in the MreB complexes, independent of the presence or absence of PBP1 (Fig. 5C, solid arrow heads) [note that the mobility of the bands in the right part (purified protein complexes) of B are slightly different from the left part (whole-cell extract), probably because of the greatly reduced protein concentrations]. A band possibly corresponding to PBP4 was also detected in the DnaC complex, although the signal was much weaker than that in the MreB complex (Fig. 5C, solid arrow head and asterisk).
The PBP1 has a molecular weight of about 99 kDa comprising a 37-amino-acid cytoplasmic N-terminus, a single 23-amino-acid transmembrane region, and an 854-amino-acid extracellular catalytic domain. Strong interactions between PBP1 with MreC and EzrA were detected previously, requiring only the transmembrane domain of PBP1 (van den Ent et al., 2006; Claessen et al., 2008). Interaction between PBP1 and GpsB was also detected previously, requiring both of the cytoplasmic tail and the transmembrane domain of PBP1 (Claessen et al., 2008; Fig. S3). As a test of whether the putative interaction between MreB and PBP1 was direct, we performed a bacterial two-hybrid experiment in E. coli host cells (which should not contain proteins sufficiently homologous or abundant to bridge the B. subtilis proteins under test). As shown in Fig. 6, MreB showed self-interaction, indicating that the fusions were functional, and a reproducible strong interaction was also detected between MreB and PBP1, although the interaction was only detectable when full-length PBP1 was coexpressed with MreB (Fig. S3). In addition, we tested whether MreB also interacted directly with other PBPs. MreB gave a positive two-hybrid signal for PBPs belonging to both class A and class B high-molecular-weight PBPs (PBP2a, PBP2b, PBP2c, PBP2d, PBP3, PBP4, PbpH and PbpI) (Fig. 6), providing support for the idea that MreB and several PBPs interact directly in the complexes detected in the pull-down experiment.
Figure 6. Bacterial two-hybrid analysis of the possible interactions between MreB, various PBPs. The T18 and T25 fragments of the adenyl cyclase protein were fused to the N-termini of MreB and several PBPs. Co-transformed strains of E. coli BTH101 expressing T25-MreB or T25 from plasmid pKT25 and T18- PBPs or T18 from pUT18C were spotted onto minimal medium supplemented with Xgal and incubated at 30°C for 48 h. The appearance of blue pigment within colonies indicates a positive interaction. The results are from a single experiment performed on the same day with strains spotted on one plate.
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