Large ring polymers align FtsZ polymers for normal septum formation
2011; Springer Nature; Volume: 30; Issue: 3 Linguagem: Inglês
10.1038/emboj.2010.345
ISSN1460-2075
AutoresMuhammet E Gündoğdu, Yoshikazu Kawai, Nad’a Pavlendová, Naotake Ogasawara, Jeff Errington, Dirk‐Jan Scheffers, Leendert W. Hamoen,
Tópico(s)Genomics and Phylogenetic Studies
ResumoArticle11 January 2011Open Access Large ring polymers align FtsZ polymers for normal septum formation Muhammet E Gündoğdu Muhammet E Gündoğdu Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Search for more papers by this author Yoshikazu Kawai Yoshikazu Kawai Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Nara Institute of Science and Technology, Graduate School of Information Science Functional Genomics, Ikoma, Japan Search for more papers by this author Nada Pavlendova Nada Pavlendova Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic Search for more papers by this author Naotake Ogasawara Naotake Ogasawara Nara Institute of Science and Technology, Graduate School of Information Science Functional Genomics, Ikoma, Japan Search for more papers by this author Jeff Errington Jeff Errington Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Search for more papers by this author Dirk-Jan Scheffers Dirk-Jan Scheffers Bacterial Membrane Proteomics Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, NN Haren, The Netherlands Search for more papers by this author Leendert W Hamoen Corresponding Author Leendert W Hamoen Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Search for more papers by this author Muhammet E Gündoğdu Muhammet E Gündoğdu Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Search for more papers by this author Yoshikazu Kawai Yoshikazu Kawai Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Nara Institute of Science and Technology, Graduate School of Information Science Functional Genomics, Ikoma, Japan Search for more papers by this author Nada Pavlendova Nada Pavlendova Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic Search for more papers by this author Naotake Ogasawara Naotake Ogasawara Nara Institute of Science and Technology, Graduate School of Information Science Functional Genomics, Ikoma, Japan Search for more papers by this author Jeff Errington Jeff Errington Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Search for more papers by this author Dirk-Jan Scheffers Dirk-Jan Scheffers Bacterial Membrane Proteomics Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, NN Haren, The Netherlands Search for more papers by this author Leendert W Hamoen Corresponding Author Leendert W Hamoen Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK Search for more papers by this author Author Information Muhammet E Gündoğdu1,‡, Yoshikazu Kawai1,2,‡, Nada Pavlendova1,3,‡, Naotake Ogasawara2, Jeff Errington1, Dirk-Jan Scheffers4,5 and Leendert W Hamoen 1 1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK 2Nara Institute of Science and Technology, Graduate School of Information Science Functional Genomics, Ikoma, Japan 3Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic 4Bacterial Membrane Proteomics Laboratory, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal 5Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, NN Haren, The Netherlands ‡These authors contributed equally to this work *Corresponding author. Institute for Cell and Molecular Biosciences, Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, Framlington Place, Newcastle NE2 4AX, UK. Tel.: +44 191 208 3240; Fax: +44 191 208 3205;E-mail: [email protected] The EMBO Journal (2011)30:617-626https://doi.org/10.1038/emboj.2010.345 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cytokinesis in bacteria is initiated by polymerization of the tubulin homologue FtsZ into a circular structure at midcell, the Z-ring. This structure functions as a scaffold for all other cell division proteins. Several proteins support assembly of the Z-ring, and one such protein, SepF, is required for normal cell division in Gram-positive bacteria and cyanobacteria. Mutation of sepF results in deformed division septa. It is unclear how SepF contributes to the synthesis of normal septa. We have studied SepF by electron microscopy (EM) and found that the protein assembles into very large (∼50 nm diameter) rings. These rings were able to bundle FtsZ protofilaments into strikingly long and regular tubular structures reminiscent of eukaryotic microtubules. SepF mutants that disturb interaction with FtsZ or that impair ring formation are no longer able to align FtsZ filaments in vitro, and fail to support normal cell division in vivo. We propose that SepF rings are required for the regular arrangement of FtsZ filaments. Absence of this ordered state could explain the grossly distorted septal morphologies seen in sepF mutants. Introduction The earliest known event in bacterial cell division is the assembly of the tubulin-like protein FtsZ into a circular structure at midcell (Adams and Errington, 2009). This so-called Z-ring recruits the other proteins needed for synthesis of the division septum. How the Z-ring is structured in the cell is not really known. Electron microscopy (EM) studies have shown that, depending on the reaction conditions, purified FtsZ can polymerize into long bundles, and structures like sheets, mini-rings, and helices (Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1999; Lu et al, 2000; Popp et al, 2009). Assembly of FtsZ into a stable Z-ring at the site of cell division involves several other proteins. One key player is FtsA, which binds FtsZ and links the Z-ring to the membrane via an amphipathic α-helical domain (Jensen et al, 2005; Pichoff and Lutkenhaus, 2005). Another conserved protein, ZapA, forms a link between FtsZ protofilaments and stimulates polymerization (Gueiros-Filho and Losick, 2002; Small et al, 2007). In rod-shaped bacteria the Min proteins exercise a regulatory role, and inhibit polymerization of FtsZ close to cell poles (Hu et al, 1999; Scheffers, 2008). Gram-positive bacteria use another FtsZ regulator, the integral membrane protein EzrA (Levin et al, 1999). Deletion of this protein leads to extra Z-rings, and it is therefore considered a negative regulator. In this study we investigated SepF (YlmF), a Z-ring-associated protein that is highly conserved in Gram-positive bacteria and cyanobacteria (Miyagishima et al, 2005; Hamoen et al, 2006; Ishikawa et al, 2006). The first indications that SepF has a role in cell division came from studies with Streptococcus pneumoniae and the cyanobacterium Synechococcus elongatus, which showed that mutations in sepF lead to severe cell division defects (Fadda et al, 2003; Miyagishima et al, 2005). Further studies with Bacillus subtilis revealed that SepF localizes to the division site. This localization depended on the presence of FtsZ, and yeast two-hybrid experiments showed a direct interaction between both proteins (Hamoen et al, 2006; Ishikawa et al, 2006). Deletion of sepF results in grossly deformed division septa in B. subtilis. Interestingly, deformed septa are not observed with other cell division mutants. SepF has an apparent functional overlap with FtsA. FtsA is not essential in B. subtilis, although B. subtilis ftsA mutants grow slower and form filamentous cells (Beall and Lutkenhaus, 1992; Jensen et al, 2005). This phenotype can be restored by overexpression of sepF. A double disruption of sepF and ftsA completely eliminates Z-ring formation and is lethal. This functional overlap with FtsA led Ishikawa et al (2006) to conclude that, like FtsA, SepF is involved in the early stages of Z-ring assembly. However, these results seem to contradict our earlier data on SepF. If SepF stimulates polymerization of FtsZ one may assume that a sepF mutant becomes sensitive for reduced FtsZ levels, but this is not the case (Hamoen et al, 2006). Furthermore, if SepF supports Z-ring formation it is unlikely that deletion of a negative regulator of FtsZ polymerization would cause problems, and yet introduction of a sepF mutation in an ezrA deletion background proved to be lethal. It is unclear at what stage of the division process SepF is active, and to gain more information on this we isolated dominant negative sepF mutants. To test whether the mutations affected the interaction with FtsZ, we purified SepF. This resulted in a rather striking observation. It turned out that SepF assembles into very large rings that can bundle FtsZ protofilaments into long and regular tubular structures. The dominant negative sepF mutants were unable to form these tubules. A mutation that blocked ring formation was also unable to align FtsZ protofilaments. The results support a new model in which SepF forms regular ring polymers that organize FtsZ protofilaments into higher order structures required for a smooth invagination of the Gram-positive septal wall. Thus, SepF is more than a simple positive regulator of Z-ring formation, which might explain why its absence is synthetic lethal in an ftsA mutant as well as in an ezrA mutant. Results SepF polymerizes into very large rings To investigate in detail how SepF influences the polymerization of FtsZ, we decided to purify both proteins for biochemical analysis. SepF was purified using maltose-binding protein (MBP) as affinity tag. The MBP moiety was cleaved from SepF and removed by ion-exchange chromatography. When subjected to size exclusion chromatography, SepF eluted in the void volume, suggesting that the protein formed aggregates. To confirm this, we examined the protein sample by EM, yet the EM images revealed a very surprising result. It proved that SepF polymerizes into very large regular ring structures with an average diameter of about 50 nm (Figure 1). These rings are so wide that, theoretically, they can encompass two whole ribosomes. The formation of these rings seemed a robust process as they were readily formed at high salt concentrations (500 mM), and in the presence of other proteins (e.g., BSA). Although the buffer used in Figure 1 contains Mg2+ (see below), this is not required for ring formation. Rings were still visible at SepF concentrations as low as 0.1 μM, but their number was greatly reduced, and they were difficult to find on the EM grid. It was therefore not possible to determine the critical concentration at which rings were formed. The ring structures display a limited variability in ring diameter (Figure 1). This suggests that SepF filaments, presumptive precursors of the closed rings, are rather inflexible and always close on themselves. Figure 1.SepF forms large ring structures. (A) EM images of negatively stained SepF rings (6 μM SepF in buffer: 50 mM Tris–HCl pH 7.4, 300 mM KCl, 10 mM MgCl2). (B) SepF rings at higher magnification. (C) Histogram showing the distribution of SepF ring diameters (213 rings counted). (D) SepF in polymerization buffer of pH 6.5 (50 mM MES pH 6.5, 50 mM KCl, 10 mM MgCl2). Scale bars: 100 nm (A, D), and 50 nm (B). Download figure Download PowerPoint SepF is an abundant protein The striking SepF rings begged the question what effect they would have on FtsZ polymerization. To analyze this under physiological conditions, we first determined the ratio of both proteins in the cell. Antibodies against purified SepF were raised and used to perform a quantitative western blot analysis. For exponentially growing cells, we estimated that the cellular concentrations of SepF and FtsZ were ∼6 and 8 μM, respectively (Supplementary Figure S1). This roughly correspond to 8000 molecules of SepF per cell, and 11 000 molecules for FtsZ, assuming a cell volume of 2.6 μm3 (Henriques et al, 1998). FtsZ is an abundant protein in the cell (Wang and Lutkenhaus, 1993), and the fact that the amount of SepF is almost as high as that of FtsZ suggests that the SepF rings have a structural role rather than a regulatory role in Z-ring formation. SepF-ring formation requires physiological reaction conditions FtsZ polymerization can be followed by pelleting and light scattering assays (Mukherjee and Lutkenhaus, 1998; Scheffers, 2008). When purified SepF was included in an FtsZ polymerization experiment a small increase in the amount of pelleted FtsZ was observed, suggesting that SepF stimulates polymerization of FtsZ (Figure 2A, lanes 1 and 2). However, we noticed a problem with the solubility of SepF. FtsZ polymerization assays are generally performed at a relatively low pH of around pH 6.5, as this stimulates the lateral association of FtsZ protofilaments (Mukherjee and Lutkenhaus, 1999). It emerged that the low pH resulted in precipitation of SepF, and EM images of SepF in a buffer of pH 6.5 showed that protein rings were no longer present (Figure 1D). Precipitation of SepF was reduced by increasing the salt concentration (300 mM KCl) and using a buffer with a more physiological pH of 7.4 (Booth, 1985; Breeuwer et al, 1996) (Supplementary Figure S2B). This effect did not depend on the type of buffer used, and the same result was obtained with a Tris, HEPES or MES buffer (data not shown). At pH 7.4 and 300 mM salt, the total amount of pelleted FtsZ was substantially less, but the stimulatory effect of SepF on pelleting was again detectable (Figure 2A, lanes 3 and 4). In light scattering assays using standard polymerization buffer of pH 6.5, B. subtilis FtsZ gave a classic response upon the addition of GTP, with a strong increase in light scatter signal that decreased over time (Supplementary Figure S3) (Mukherjee and Lutkenhaus, 1999). The light scattering signal was strongly reduced by a shift to pH 7.4, and almost eliminated by increased salt concentrations (Supplementary Figure S3), indicating that lateral interactions between FtsZ protofilaments are greatly diminished under these conditions. The addition of SepF did result in a shift in light scattering signal (Figure 2B) but not the gradual rise that is normally associated with bundling of FtsZ protofilaments. SepF had no measurable effect on the GTPase activity of FtsZ (Figure 2C). Figure 2.Biochemical analyses of the effect of SepF on FtsZ polymerization. (A) Results from three independent FtsZ sedimentation experiments. The increase in pelleting after addition of GTP (lanes 1 and 3) or GTP and SepF (lanes 2 and 4) are shown (see Supplementary Figure S2A for SDS–PAA gel). Lanes 1 and 2 are samples prepared in pH 6.5 polymerization buffer (50 mM MES pH 6.5, 50 mM KCl, 10 mM MgCl2), and lanes 3 and 4 are samples prepared in pH 7.4 buffer (50 mM Tris–HCl pH 7.4, 300 mM KCl, 10 mM MgCl2). (B) Light scattering analyses of FtsZ polymerization in pH 7.4 buffer. FtsZ (a), SepF (b), and GTP (c) were added in subsequent steps. (C) GTP hydrolysis during FtsZ polymerization in pH 7.4 buffer (average of four independent experiments). In all experiments 1 mM GTP, 10 μM FtsZ, and 6 μM SepF were used. Download figure Download PowerPoint SepF bundles FtsZ into tubules We returned to EM to investigate whether the SepF rings had any effect on FtsZ filaments. In a buffer of pH 7.4 with 300 mM KCl, FtsZ forms clear protofilaments when GTP is present (Figure 3A). The addition of SepF had a remarkable result and long regular tubular structures became visible (Figure 3B). The average diameter of these structures was about 48 nm (Figure 3C), close to the diameter of SepF rings. Tubule formation was not influenced by the order in which the proteins or GTP were added, but depended on the concentration of FtsZ. When half the amount of FtsZ was used (5 μM) less tubules were detected. Increasing the SepF concentration did not compensate for this. At half the SepF concentration (3 μM) tubules were still visible, but at 1 μM SepF and 10 μM FtsZ no tubules could be identified on the EM grids. When GDP instead of GTP was used no tubules were formed. We never observed tubules in polymerization buffer of pH 6.5, which may explain why they were not observed in a previous study (Singh et al, 2008). We used 10 mM Mg2+ in the polymerization buffers, which is commonly used for FtsZ polymerization studies, but 1 mM Mg2+ was sufficient to see tubules. Without Mg2+ no tubules were formed. At higher magnifications the tubules appear to consist of straight longitudinal filaments, most likely FtsZ polymers (Figure 3D, narrow arrows), with evident transverse bands that presumably represent SepF rings (Figure 3D, wide arrows). From the EM images it seems that the SepF rings form the core of the FtsZ–SepF tubules, but attempts to resolve this question using thin section EM or Cryo-EM were unsuccessful. The length of the tubules increased with time and tubules could grow up to micrometres in length (Figure 3E; Supplementary Figure S4). Interestingly, during the first 5 min of the reaction filaments emanating from the ends of the short tubules tended to be straight (Figure 4A and B), whereas after 10 min, some tubules had splayed tips with highly curved filaments (Figure 4C–E). In the later samples (20 min), tubules with different kind of bifurcations, including branches and rings, were evident (Figures 3E and 4F–I). Figure 3.EM images of negatively stained FtsZ tubules formed by SepF rings. (A) FtsZ protofilaments in polymerization buffer pH 7.4 after the addition of 1 mM GTP and (B) under the same conditions in the presence of SepF. Concentrations used for FtsZ and SepF were 10 and 6 μM, respectively. (C) Histogram showing the distribution of FtsZ–SepF tubule widths (148 measurements). (D) Detailed picture of two FtsZ–SepF tubules. In the upper tubule SepF rings are indicated by arrowheads, and in the lower tubule longitudinal FtsZ filaments are indicated by arrowheads. (E) FtsZ–SepF tubules can grow up to a few micrometres in length. Scale bars: 100 nm (A, B), 50 nm (D), and 500 nm (E). Download figure Download PowerPoint Figure 4.Compilation of EM images showing FtsZ–SepF tubule ends at different time points in the reaction. (A, B) Tubule ends after 5 min showing relative straight filaments. (C–E) Curved ends are observed after about 10 min. (F, G) Some tubule ends show bifurcations after 20 min incubation. (H, I) Detail of branching FtsZ–SepF tubules. Scale bar: 100 nm. Download figure Download PowerPoint Screen for SepF mutations that inhibit cell division Although the formation of FtsZ–SepF tubules appeared a reproducible and robust process in vitro, it was important to test its physiological relevance. We investigated whether it was possible to isolate dominant negative mutations in SepF that would affect the interaction of SepF with FtsZ and so disrupt tubule formation. The screen took advantage of the fact that a combination of sepF and ftsA null mutations is lethal (Ishikawa et al, 2006). Randomly mutagenized alleles of sepF were expressed from a xylose inducible Pxyl-promoter in cells carrying a wild-type sepF gene and a deletion of ftsA. After testing several thousands of transformants two clones were identified that were unable to grow when the sepF mutant allele was overexpressed by the addition of xylose to the growth medium (Figure 5A). Sequencing of the mutated sepF alleles revealed two different mutations affecting conserved residues: A98V and F124S (Figure 6). When these SepF mutants were (over)expressed in a strain devoid of wild-type SepF, cells remained elongated, and there was no indication that the mutant proteins could compensate for the absence of wild-type protein. To test whether the dominant negative sepF mutants affected Z-ring formation, we examined the localization of a gfp–ftsZ fusion. As shown in Figure 5B (upper panel), the overexpression of wild-type SepF in an ftsA mutant background had no effect on Z-ring formation. However, when the mutants A98V or F124S were induced (Figure 5B, lower panels), Z-ring formation was abolished, indicating that the mutant proteins inhibit cell division by interfering with Z-ring formation. Figure 5.Isolation of SepF mutations that inhibit cell division. (A) The isolated mutations A98V and F124S were lethal when expressed (+xylose) in a ΔftsA background. Strains were plated in the presence or absence of 2% xylose. The background genotypes are indicated above the plates. (B) Localization of GFP–FtsZ after expression of the SepF mutants (1% xylose) in a ΔftsA background (deletion of ftsA reduces cell division and leads to elongated cells). Scale bar: 5 μm. Download figure Download PowerPoint Figure 6.Amino acid sequence alignment of SepF proteins, from B. subtilis (Bsub), Listeria monocytogenes (Lmon), Staphylococcus aureus (Saur), and Streptomyces coelicolor (Scoe), using ClustalW. Secondary structure prediction (using PSIPRED) for B. subtilis SepF is shown above the sequences. The position of α-helices and β-sheets are indicated by rods and arrows, respectively. Conserved amino acids are marked in grey, and the mutated amino acids are indicated above the sequence (A98V, F124S, and G135N). In mutant Δ134, the protein is truncated at amino acid 134. Download figure Download PowerPoint Dominant negative SepF mutants do not form tubules The inability of the SepF mutants to support growth could be a consequence of mutated FtsZ-binding sites or possibly a failure to form ring structures. To test this, the mutant SepF proteins were purified and first analyzed by size exclusion chromatography. Wild-type SepF showed a large peak in the void volume (>2 MDa), probably corresponding to SepF rings (Figure 7A). The F124S mutant showed a comparable elution profile to the wild-type protein. However, the A98V mutant was almost absent from the void volume, suggesting that this mutant affects ring formation. This protein showed an increased elution at around 14 ml between the peaks for the reference proteins thyroglobulin (669 kDa) and aldolase (158 kDa). Thus, the A98V mutation still gives oligomeric structures and does not reduce the protein to its monomeric state of 17 kDa. EM analysis showed normal ring formation for the F124S mutant (Figure 7B). In case of the A98V mutant it was difficult to find rings on the EM grids, indicating that this mutant protein is indeed disturbed in ring formation. Figure 7.Effects of mutations on ring formation and FtsZ binding. (A) Size exclusion chromatography of the different SepF mutants using an analytical Superose 6 column. The absorbance at 280 nm (AU) is plotted as a function of the elution volume. The peaks of two reference proteins thyroglobulin (T, 669 kDa) and aldolase (A, 158 kDa) are indicated by arrows. SepF has a calculated size of 17 kDa. (B) EM images of negatively stained SepF mutants. Scale bar is 50 nm (buffer used; 20 mM Tris–HCl pH 7.4, 200 mM KCl, 5 mM MgCl2). (C) Western blot analyses of a SepF–FtsZ co-elution experiment using different MBP–SepF mutants. The columns were incubated with (+) or without (−) B. subtilis FtsZ. FtsZ-antiserum was used to stain the blots (see Materials and methods, for details). Download figure Download PowerPoint We then examined the ability of the mutant proteins to support the formation of FtsZ tubules. Importantly, neither of the mutant proteins formed any detectable tubular structures under conditions in which wild-type SepF supported abundant tubulation (Supplementary Figure S5). As the F124S protein still forms rings, it is possible that this mutant is unable to interact with FtsZ. To test this, we performed an FtsZ–SepF co-elution experiment using MBP–SepF fusions. MBP–SepF, when bound to amylose resin, is capable of selectively binding FtsZ from an extract of Escherichia coli cells expressing B. subtilis FtsZ (Figures 7C and 8A). MBP–SepF mutant fusions, bound to amylose resin, retained significantly less FtsZ compared with wild-type protein. In fact, the amount of FtsZ that interacted with these mutant proteins was only detectable by western blot analysis. Thus, both A98V and F124S mutations impair the interaction with FtsZ protein. GFP fusions with these SepF mutants showed diffuse fluorescence in cells, in agreement with a loss of FtsZ-binding activity (data not shown). Figure 8.Characteristics of a SepF mutant defective in ring formation. (A) SepF–FtsZ co-elution experiment using different MBP–SepF mutants. The elution fractions where analyzed by SDS–PAGE and Coomassie staining. The columns were incubated with (+) or without (−) B. subtilis FtsZ. As a negative control A98V and F124S were included. (B) Localization of SepF–G135N–GFP fusion in ΔsepF B. subtilis cells. The fusion protein (white bands) is located at cell division sites. (C) Images of negatively stained purified SepF–G135N (left panel), and in the presence of FtsZ (right panel, FtsZ protofilaments are clearly visible). Scale bar: 200 nm. (D) Mutation G135N is unable to sustain growth in the absence of FtsA. (E) Western blot analysis of SepF levels in wild-type B. subtilis cells (wt), a sepF knockout strain (ΔsepF), and a strain expressing SepF–G135N (G135N). Download figure Download PowerPoint The F124S mutant largely retained the ability to form rings, and it is likely that its phenotype is manifested mainly through impaired interaction with FtsZ. Possibly, this protein interferes with wild-type SepF by creating hybrid rings that are less efficient in bundling FtsZ protofilaments. The A98V mutant could have a similar effect. We have tried to visualize hybrid rings by employing SepF fusion proteins, such as an N-terminal MBP–SepF fusion or a C-terminal SepF–Intein fusion, but both fusion proteins were unable to form rings by themselves. This could also explain why GFP fusions of SepF are not active in vivo. Indeed, when we examined purified SepF–GFP by EM no protein rings were detected (data not shown). Breaking the ring abolishes tubule formation The above results suggest that alignment of FtsZ protofilaments is a key activity of SepF. However, it is still unclear whether the formation of rings is important for this activity. The inability of a SepF–GFP fusion to form rings indicates that the C-terminus is probably important for ring formation. Indeed, removal of the putative α-helical domain at the C-terminus (mutant Δ134) abolishes formation of SepF rings and of SepF–FtsZ tubules, even though the truncated SepF can still bind FtsZ (Figure 8A, data not shown). The first residue in this 15 amino acid C-terminal domain is a conserved glycine (Figure 6). Mutation of this glycine to asparagine (G135N) had no consequences for SepF–FtsZ interaction as the mutant protein still bound FtsZ in vitro (Figure 8A), and localized to the Z-ring in vivo (Figure 8B). When we analyzed purified G135N mutant protein by EM no protein rings were detectable, instead the protein appeared to assemble into long filamentous structures (Figure 8C, left panel). Importantly, mixing the mutant protein with purified FtsZ did not result in tubular structures (Figure 8C, right panel). These data suggest that the C-terminus of SepF is required for the formation of rings, and that SepF rings are required for the alignment of FtsZ polymers. To test whether the sepF-ring mutant was active in vivo, we replaced wild-type sepF with sepF–G135N in a B. subtilis strain that contains ftsA under control of the IPTG-inducible Pspac promoter. When the resulting strain was grown on plates without IPTG, no colonies appeared (Figure 8D), despite the fact that expression levels of SepF–G135N were normal (Figure 8E). The mutant, when overexpressed in an ftsA deletion strain, showed no dominant negative effect like the SepF mutants A98V and F124S. The finding that SepF–G135N cannot support cell growth in the absence of FtsA indicates that SepF-ring formation is critical for the function of SepF, and lends further support to the conclusion that the organization of FtsZ protofilaments by SepF rings is important for correct cell division. Discussion This is the first report of a protein polymer that directly supports bundling of FtsZ polymers. Even more surprising is the fact that this polymer closes into a ring. There are other cell division proteins that have been shown to promote bundling of FtsZ filaments, including ZapA of B. subtilis and E. coli (Gueiros-Filho and Losick, 2002; Small et al, 2007), and the E. coli protein ZipA (RayChaudhuri, 1999; Hale et al, 2000), but the FtsZ bundles formed by these proteins do not display
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