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Filament Formation of the FtsZ/Tubulin-like Protein TubZ from the Bacillus cereus pXO1 Plasmid*

  • Shota Hoshino
    Affiliations
    Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045, Japan
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  • Ikuko Hayashi
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045, Japan
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  • Author Footnotes
    * This work was supported by the Hayashi Memorial Foundation for Female Natural Scientists (to I. H.).
    This article contains supplemental “Experimental Procedures,” Figs. S1–S8, and an additional reference.
Open AccessPublished:July 30, 2012DOI:https://doi.org/10.1074/jbc.M112.373803
      Stable maintenance of low-copy-number plasmids requires partition (par) systems that consist of a nucleotide hydrolase, a DNA-binding protein, and a cis-acting DNA-binding site. The FtsZ/tubulin-like GTPase TubZ was identified as a partitioning factor of the virulence plasmids pBtoxis and pXO1 in Bacillus thuringiensis and Bacillus anthracis, respectively. TubZ exhibits high GTPase activity and assembles into polymers both in vivo and in vitro, and its “treadmilling” movement is required for plasmid stability in the cell. To investigate the molecular mechanism of pXO1 plasmid segregation by TubZ filaments, we determined the crystal structures of Bacillus cereus TubZ in apo-, GDP-, and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-bound forms at resolutions of 2.1, 1.9, and 3.3 Å, respectively. Interestingly, the slowly hydrolyzable GTP analog GTPγS was hydrolyzed to GDP in the crystal. In the post-GTP hydrolysis state, GDP-bound B. cereus TubZ forms a dimer by the head-to-tail association of individual subunits in the asymmetric unit, which is similar to the protofilament formation of FtsZ and B. thuringiensis TubZ. However, the M loop interacts with the nucleotide-binding site of the adjacent subunit and stabilizes the filament structure in a different manner, which indicates that the molecular assembly of the TubZ-related par systems is not stringently conserved. Furthermore, we show that the C-terminal tail of TubZ is required for association with the DNA-binding protein TubR. Using a combination of crystallography, site-directed mutagenesis, and biochemical analysis, our results provide the structural basis of the TubZ polymer that may drive DNA segregation.

      Introduction

      The cytoskeleton is a cellular scaffold made up of protein filaments. The dynamic network of the filaments is essential for many biological phenomena, such as cell division, chromosomal segregation, and cell movement. Until recently, it was thought that the cytoskeleton was a feature unique to eukaryotes. Analyses of cells using immunofluorescence and live-cell fluorescence microscopy have shown that filamentous structures indeed exist in prokaryotic cells, although their dynamics differ greatly (reviewed in Refs.
      • Carballido-López R.
      • Errington J.
      A dynamic bacterial cytoskeleton.
      ,
      • Graumann P.L.
      Cytoskeletal elements in bacteria.
      ,
      • Cabeen M.T.
      • Jacobs-Wagner C.
      The bacterial cytoskeleton.
      ). FtsZ, the bacterial homolog of tubulin, is highly conserved in eubacteria and archaea (
      • Makarova K.S.
      • Koonin E.V.
      Two new families of the FtsZ/tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea.
      ). Whereas tubulin is a major component of the mitotic spindle and plays a vital role in chromosomal segregation, FtsZ forms a contractile Z-ring at midcell and drives cytokinesis, which is functionally analogous to the actin contractile ring (
      • van den Ent F.
      • Amos L.
      • Löwe J.
      Bacterial ancestry of actin and tubulin.
      ). Although FtsZ is evolutionally distant from tubulin, they both assemble into polymers in a GTP-dependent manner (
      • Erickson H.P.
      • Taylor D.W.
      • Taylor K.A.
      • Bramhill D.
      Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.
      ,
      • Weisenberg R.C.
      • Deery W.J.
      • Dickinson P.J.
      Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules.
      ). The similarity of their three-dimensional structures further provides evidence that they evolved from a common ancestor (
      • Löwe J.
      • Amos L.A.
      Crystal structure of the bacterial cell division protein FtsZ.
      ,
      • Nogales E.
      • Downing K.H.
      • Amos L.A.
      • Löwe J.
      Tubulin and FtsZ form a distinct family of GTPases.
      ).
      Bacterial low-copy-number plasmids generally encode par genes to achieve proper DNA segregation into daughter cells. The par system consists of three components: a centromeric DNA site, a centromere-binding protein, and a nucleotide hydrolase, which acts as a linear motor (
      • Ebersbach G.
      • Gerdes K.
      Plasmid segregation mechanisms.
      ). By classifying the hydrolase components, the systems fall into three categories because the hydrolases are more distinct than the centromere-binding proteins. Type I partition systems use ParA ATPase proteins with a Walker-type motif, whereas type II systems use actin-like ATPases called ParM. Recently, a new partition system has been identified in virulent Bacillus species (
      • Tinsley E.
      • Khan S.A.
      A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis.
      ,
      • Tang M.
      • Bideshi D.K.
      • Park H.W.
      • Federici B.A.
      Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis.
      ). The system involves an FtsZ/tubulin-like protein called TubZ or RepX, which is essential for the maintenance of the pBtoxis plasmid from Bacillus thuringiensis (Bt)
      The abbreviations used are: Bt
      B. thuringiensis
      Ba
      B. anthracis
      Bc
      B. cereus
      Cb
      C. botulinum
      GTPγS
      guanosine 5′-3-O-(thio)triphosphate
      BisTris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
      and the pXO1 plasmid from Bacillus anthracis (Ba) and Bacillus cereus (Bc) (hereafter named Bt-TubZ, Ba-TubZ, and Bc-TubZ, respectively). TubZ polymerizes in a GTP-dependent manner in vitro and treadmills in vivo, i.e. the plus-end of a filament grows in length, whereas the minus-end shrinks, resulting in directional polymerization (
      • Tang M.
      • Bideshi D.K.
      • Park H.W.
      • Federici B.A.
      Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis.
      ,
      • Chen Y.
      • Erickson H.P.
      In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.
      ,
      • Larsen R.A.
      • Cusumano C.
      • Fujioka A.
      • Lim-Fong G.
      • Patterson P.
      • Pogliano J.
      Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.
      ). A point mutation that eliminates TubZ GTP hydrolysis negatively affects plasmid stability, which suggests that TubZ is one of the tubulin-based cytoskeletal proteins whose polymer acts as a “cytomotive” filament in the force-generating system to accomplish plasmid partitioning (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ). The tubZ gene is co-transcribed with tubR, a gene that encodes a DNA-binding protein in the tubRZ operon. Genetic analyses of the minimal replicon of the pBtoxis and pXO1 plasmids showed that the tubRZ operon is essential for maintaining the plasmids in host cells (
      • Tinsley E.
      • Khan S.A.
      A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis.
      ,
      • Tang M.
      • Bideshi D.K.
      • Park H.W.
      • Federici B.A.
      Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis.
      ). In B. thuringiensis, TubR (Bt-TubR) binds to the promoter region of the tubRZ operon, negatively regulates its transcription, and presumably recruits TubZ to the partition site (
      • Tang M.
      • Bideshi D.K.
      • Park H.W.
      • Federici B.A.
      Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis.
      ,
      • Larsen R.A.
      • Cusumano C.
      • Fujioka A.
      • Lim-Fong G.
      • Patterson P.
      • Pogliano J.
      Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.
      ,
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ). Although the gene organization of the tubRZ operon is similar among virulent bacilli, Ba-TubZ and Bc-TubZ, which are 98% identical, are quite divergent from Bt-TubZ, sharing only 21% amino acid identity (
      • Makarova K.S.
      • Koonin E.V.
      Two new families of the FtsZ/tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea.
      ,
      • Chen Y.
      • Erickson H.P.
      In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.
      ). Recently, the crystal structures of Bt-TubZ were determined, revealing that it belongs to the FtsZ/tubulin superfamily of proteins (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ,
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). Electron microscopy studies showed that, unlike FtsZ or tubulin, Bt-TubZ forms a double helical filament similar to the polymer structure of the actin-like ATPase ParM (
      • Chen Y.
      • Erickson H.P.
      In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.
      ,
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). To elucidate the detailed mechanism of Bc-TubZ filament formation, we determined the crystal structures of Bc-TubZ in the apo-, GDP-, and GTPγS-bound forms. Together with biochemical analysis, we found that the M loop mediates filament stabilization in Bc-TubZ, which suggests that diverse mechanisms facilitate molecular recognition in filament formation for plasmid segregation.

      EXPERIMENTAL PROCEDURES

      Cloning, Protein Expression, and Purification

      The tubZ gene from B. cereus ATCC 10987 (Bacillus Genetic Stock Center) was amplified by PCR from genomic DNA and cloned into pET21d with or without a stop codon for the wild-type protein and the protein containing a C-terminal histidine tag, respectively. All proteins were expressed in Escherichia coli strain BL21(DE3). Wild-type protein was purified from the bacterial extract by 40% saturated ammonium sulfate precipitation, followed by HiTrap Q ion exchange chromatography (GE Healthcare). Histidine-tagged proteins were purified with HisTrap HP, followed by ion exchange and size exclusion chromatographies using Resource Q and Superdex 200 (GE Healthcare), respectively.
      The Bc-TubZ protein with a C-terminal truncation (Bc-TubZΔ, residues 1–389) was characterized by limited proteolysis of wild-type Bc-TubZ with the thermolysin protease. The protein fragment was analyzed by N-terminal sequencing and mass spectroscopy. The Bc-tubZΔ and Bc-tubR genes were cloned into pET28a using NdeI and NotI in-frame with an N-terminal histidine tag and an additional tobacco etch virus protease recognition sequence. After cleavage of the tag by the histidine-tagged tobacco etch virus protease, the protein was loaded onto a Resource Q column (
      • Blommel P.G.
      • Fox B.G.
      A combined approach to improving large-scale production of tobacco etch virus protease.
      ). Fractions containing Bc-TubZΔ were pooled and concentrated to 0.5 mm for crystallization. Selenomethionine-substituted Bc-TubZΔ was expressed in BL21(DE3) cells as described previously and purified in the same manner as the wild-type protein (
      • Van Duyne G.D.
      • Standaert R.F.
      • Karplus P.A.
      • Schreiber S.L.
      • Clardy J.
      Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin.
      ). Amino acid mutations were introduced by PCR-based site-directed mutagenesis. The DNA fragment in pBc10987 containing the tubRZ operon (nucleotides 64822–69262) was cloned into pBlueScriptSK using XbaI and XhoI sites (pBS_Bc-tubRZ). For all of the constructs, the absence of error was confirmed by DNA sequencing.

      Crystallization, Data Collection, and Structure Determination

      Crystals of selenomethionine-labeled apo-Bc-TubZΔ were obtained by hanging drop vapor diffusion at 20 °C with reservoir solution containing 0.1 m BisTris (pH 5.6), 0.2 m MgCl2, and 30% PEG 4000. Crystals of GDP-Bc-TubZΔ appeared after 2–3 weeks in 0.1 m MES (pH 5.5), 1 mm MgCl2, and 50% PEG 400 in the presence of 1 mm GTPγS. Crystals of the GTPγS-bound form were obtained by soaking 2 mm GTPγS into the GDP-Bc-TubZΔ crystal for 30 min. All crystals were cryoprotected with 25% glycerol in the mother liquor.
      Diffraction images were processed with HKL2000 (
      • Otwinowski Z.
      • Minor W.
      Processing of x-ray diffraction data collected in oscillation mode.
      ). The initial phases for apo-Bc-TubZΔ were obtained by selenium multiwavelength anomalous diffraction with the PHENIX program (AutoSol) (
      • Adams P.D.
      • Grosse-Kunstleve R.W.
      • Hung L.W.
      • Ioerger T.R.
      • McCoy A.J.
      • Moriarty N.W.
      • Read R.J.
      • Sacchettini J.C.
      • Sauter N.K.
      • Terwilliger T.C.
      PHENIX: building new software for automated crystallographic structure determination.
      ) (Table 1). After automatic model building, the remaining residues were built manually in Coot (
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      ). Both GDP-Bc-TubZΔ and GTPγS-Bc-TubZΔ structures were solved by molecular replacement using Phaser with the apo-Bc-TubZΔ structure (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ). The structures were refined using CNS (
      • Brünger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      Crystallography & NMR system: a new software suite for macromolecular structure determination.
      ).
      TABLE 1Crystallographic statistics
      Apo-Bc-TubZ (Se-MAD)GDP-Bc-TubZGTPγS-Bc-TubZ
      Data collection
      BeamlineNW12A (Photon Factory)BL26B2 (SPring-8)BL26B2 (Spring-8)
      Space groupP21P21P21
      Unit cell dimensions (a, b, c; β)54.8, 66.0, 58.4 Å; 106.9°48.8, 76.0, 96.9 Å; 104.6°49.5, 76.4, 97.6 Å; 105.2°
      Data range (Å)50–2.150–1.950–3.3
      PeakEdgeHigh remote
      Wavelength (Å)0.979210.979400.964171.00001.0000
      No. unique reflections45,58745,60745,81752,86210,758
      Completeness (%)99.7 (99.9)99.7 (99.9)99.8 (99.7)97.8 (96.9)99.8 (100.0)
      Multiplicity3.9 (3.8)3.8 (3.8)3.8 (3.7)5.2 (5.0)7.0 (6.8)
      I/σ(I)20.5 (5.5)19.9 (4.78)16.4 (2.59)23.3 (3.5)22.7 (4.5)
      Rmerge
      Rmerge = Σ|Iobs − 〈I〉|/ΣIobs, where Iobs is the intensity measurement and 〈I〉 is the mean intensity for multiply recorded reflections (20).
      0.049 (0.224)0.050 (0.278)0.062 (0.471)0.059 (0.441)0.082 (0.377)
      Overall figure of merit0.53 (SOLVE), 0.91 (RESOLVE)
      Refinement
      Resolution range (Å)50–2.150–1.950–3.3
      No. reflections in working set39,98545,9129285
      Rcryst (Rfree)
      Rcryst and Rfree = ΣFobs| − |Fcalc‖/|Fobs| for reflections in the working and test sets, respectively. The Rfree value was calculated using a randomly selected 10% of the data set that was omitted through all stages of refinement.
      0.199 (0.247)0.185 (0.237)0.206 (0.294)
      R.M.S.D. bond length (Å)0.00890.00780.0030
      R.M.S.D. bond angle1.4°1.4°0.76°
      Most favored area (%)94.597.088.7
      Additionally allowed area (%)3.52.810.1
      Generously allowed area (%)2.000
      Disallowed area (%)00.21.2
      PDB code4EI84EI74EI9
      a Rmerge = Σ|Iobs − 〈I〉|/ΣIobs, where Iobs is the intensity measurement and 〈I〉 is the mean intensity for multiply recorded reflections (
      • Otwinowski Z.
      • Minor W.
      Processing of x-ray diffraction data collected in oscillation mode.
      ).
      b Rcryst and Rfree = ΣFobs| − |Fcalc‖/|Fobs| for reflections in the working and test sets, respectively. The Rfree value was calculated using a randomly selected 10% of the data set that was omitted through all stages of refinement.

      Bc-TubZ Polymerization and GTPase Analyses

      Analyses of Bc-TubZ polymerization and GTPase activity were performed as described (
      • Chen Y.
      • Erickson H.P.
      In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.
      ). Briefly, Bc-TubZ was polymerized at 35 °C in HMK100 buffer (50 mm HEPES (pH 7.7), 100 mm KAc, 5 mm MgAc, and 1 mm EGTA) with 0.5 mm GTP and 5 μm GTPγS. The turbidity of 2 μm Bc-TubZ was recorded by 90° angle light scattering using a Shimadzu RF-5300PC spectrofluorometer with both the excitation and emission wavelengths set at 360 nm. For GTPase activity analyses, 2 μm Bc-TubZ was polymerized in HMK100 buffer with 0.4 mm phosphoenolpyruvate, 0.3 mm NADH, 20 units/ml pyruvate kinase, 20 units/ml lactate dehydrogenase, and 0.5 mm GTP. NADH concentration was monitored by absorbance at 340 nm using a Jasco V-630BIO UV-visible photometer. The effects of Bc-TubR and DNA were examined by the addition of 2 μm Bc-TubR and 0.3 nm pBS_Bc-tubRZ to the reaction mixture.

      TubZ Sedimentation

      Bc-TubZ and Bc-TubZ-His (40 μm) were assembled in HMK100 buffer with 0.1 mm GTP and 1 μm GTPγS for 10 min at 35 °C in a 50-μl reaction volume. After incubation, 5 μg of Bc-TubR was added and incubated for 5 min at 35 °C. The reaction mixtures were layered onto a 12% glycerol cushion with 0.1 mm GTP in HMK100 buffer and centrifuged using a Beckman TLA-100.3 rotor at 80,000 rpm for 30 min. Pellets were resuspended in the loading buffer and analyzed by 15% SDS-PAGE with Coomassie Blue staining.

      RESULTS

      Overall Structure of Bc-TubZ

      Although recombinant Bc-TubZ was expressed at high levels, crystallization trials of wild-type Bc-TubZ failed. Because Bc-TubZ was susceptible to proteolysis during purification, we proteolytically digested Bc-TubZ and identified the stable fragment. The truncated fragment Bc-TubZΔ (residues 1–389) was expressed as a fusion protein with an N-terminal histidine tag, purified to homogeneity, and crystallized with or without GTPγS. The apo-Bc-TubZΔ structure was determined by multiwavelength anomalous diffraction. Although crystallization screening for the GTP-bound form of Bc-TubZΔ was performed in the presence of the slowly hydrolyzable GTP analog GTPγS, we were only able to obtain crystals of Bc-TubZΔ in complex with GDP, as judged by the FoFc omit electron density map (supplemental Fig. S1). The structure of GTPγS-Bc-TubZΔ was only obtained by soaking GTPγS into GDP-Bc-TubZΔ crystals. The structures of both GTPγS-Bc-TubZΔ and GDP-Bc-TubZΔ were solved by molecular replacement using the apo-Bc-TubZΔ structure as a search model. The model of apo-Bc-TubZΔ consists of residues 3–60, 62–63, 68–79, and 89–376 (Fig. 1A). The asymmetric unit contains a monomer that has two functional domains, an N-terminal GTP-binding domain and a C-terminal GTPase-activating domain, as seen in the FtsZ structure (
      • Erickson H.P.
      Atomic structures of tubulin and FtsZ.
      ,
      • Oliva M.A.
      • Cordell S.C.
      • Löwe J.
      Structural insights into FtsZ protofilament formation.
      ). Crystallographic packing and gel filtration analyses showed that apo-Bc-TubZΔ is monomeric (supplemental Fig. S2). Three regions adjacent to the guanine nucleotide-binding pocket (residues 61 and 64–67 in the T2 loop and residues 80–88 in the T3 loop) (supplemental Fig. S3) are disordered and exhibit no clear electron density. The base recognition loop T5 is positioned above the γ-phosphate contact T4 with the signature sequence of the FtsZ/TubZ family proteins (122GGGTGTG128) and reveals higher temperature factors of 57.6 Å2 (35.0 Å2 for the overall structure), indicating that the guanine base is tightly recognized by the T5 loop. When the protein binds to the nucleotide, the corresponding regions become well ordered and form hydrogen bonds to the nucleotide (Fig. 1, B and C). Furthermore, the conformational change occurs within the C-terminal portion of Bc-TubZΔ and its neighboring helix H5 (Fig. 1D). In the apo-form, the H5 helix region is partially unfolded and makes a kink at Ala-163, thus contacting H11 in a parallel manner by forming hydrophobic interactions among Leu-157 and Leu-160 in H5 and Val-366 and Ile-369 in H11 (supplemental Fig. S2C). On the other hand, H11 in the nucleotide-bound form pivots around Leu-362 by 50°, which leads H11 to contact H5 in an antiparallel manner (Fig. 1E). Lys-377 in H11 forms a hydrogen bond with Glu-161 in H5. The surrounding residues, Leu-157, Leu-160, Ile-369, Ile-373, and Ile-376, create a hydrophobic core that stabilizes the interaction. We presume that this conformational change is caused by the structural instability of apo-Bc-TubZΔ because the structures of apo-Bt-TubZ and GDP-Bc-TubZΔ are identical, despite a lack of primary sequence similarity (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ). Although the C terminus of Bc-TubZ is proteolytically sensitive and is suggested to be locally unfolded, the intramolecular interaction of the tail seems to stabilize Bc-TubZ because the melting points of Bc-TubZ and Bc-TubZΔ are 59.0 and 54.8 °C, respectively (supplemental Fig. S4). The GDP-Bc-TubZΔ crystal structure reveals two molecules in the asymmetric unit, which is semicontinuous in the crystal lattice (see below). H11 of GDP-Bc-TubZΔ is directed toward the nearest longitudinal subunit. Nucleotide binding induces the conformational change of H5, by which H11 may convey an activating signal of Bc-TubZ polymerization to the adjacent subunit. In the GTPγS-bound structure, one of the GDP molecules is replaced with GTPγS in the same nucleotide-binding pocket, which induces no conformational change in the vicinity of GTPγS (supplemental Fig. S1).
      Figure thumbnail gr1
      FIGURE 1Crystal structure of Bc-TubZ. A and B, schematic representations of apo-Bc-TubZΔ and GDP-Bc-TubZΔ, respectively. α-Helices are colored green, and β-strands are colored orange. Secondary structural elements are labeled in A. The active site loops (T1–T7) and loops on the other side of the molecule (M, T0, and T7) are labeled in B. GDP is shown as a space-filling model. C, close-up view of the active site in the GDP-bound form. The nucleotide recognition loops are colored cyan. Interacting residues are shown as sticks. D, conformational change of H11. The apo-bound form is colored yellow, and the nucleotide-bound form is colored green. The hinge residue Leu-362 is labeled (red arrowhead). E, stereo view of the interaction between H5 and H11. Residues involved in the interaction are labeled and shown as sticks (Glu-161 in magenta, Lys-377 in blue, and hydrophobic residues in yellow). This figure was made using PyMOL (
      • DeLano W.L.
      ).

      Structural Comparison of TubZ and FtsZ Proteins

      To date, the crystal structures of Bt-TubZ and Clostridium botulinum phage C strain Stockholm TubZ (Cb-TubZ) have been determined (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ,
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ,
      • Oliva M.A.
      • Martin-Galiano A.J.
      • Sakaguchi Y.
      • Andreu J.M.
      Tubulin homolog TubZ in a phage-encoded partition system.
      ). Superposition of GTPγS-Bc-TubZ with GTPγS-Bt-TubZ and apo-Cb-TubZ shows that their structures resemble one another (root mean square deviation of 2.3 Å in 326 Cα positions and 2.5 Å in 291 Cα positions, respectively) (Fig. 2A). The N-terminal domain is structurally well conserved among the FtsZ/tubulin family proteins and is related to typical GTPases with a Rossmann fold, such as p21ras (
      • Löwe J.
      • Amos L.A.
      Crystal structure of the bacterial cell division protein FtsZ.
      ). The GTPase domain of Bc-TubZ (residues 1–200) shares relatively low sequence homology (25%) with the N terminus of Methanococcus jannaschii FtsZ but displays significant structural similarity, with a root mean square deviation of 2.5 Å over 170 Cα atoms (Fig. 2B). Notably, although B. cereus and B. thuringiensis are closely related species that share a similar genetic background, the GTPase domains from Bc-TubZ and Bt-TubZ show weak sequence conservation (23%), indicating that Bc-TubZ is evolutionally divergent from Bt-TubZ to a similar extent as FtsZ (
      • Makarova K.S.
      • Koonin E.V.
      Two new families of the FtsZ/tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea.
      ,
      • Helgason E.
      • Okstad O.A.
      • Caugant D.A.
      • Johansen H.A.
      • Fouet A.
      • Mock M.
      • Hegna I.
      • Kolstø A.B.
      Bacillus anthracis Bacillus cereus Bacillus thuringiensis–one species on the basis of genetic evidence.
      ). The GTPase domain of Cb-TubZ shares only 21% sequence identity with that of Bc-TubZ and has a remarkably truncated H6 helix. Furthermore, Cb-TubZ lacks H0, which locates between the N- and C-terminal domains of both Bc-TubZ and Bt-TubZ. The C-terminal domain of the FtsZ/tubulin family proteins is less conserved but has an essential role in the activation of GTP hydrolysis (
      • Oliva M.A.
      • Cordell S.C.
      • Löwe J.
      Structural insights into FtsZ protofilament formation.
      ). The superposition between the GTPase-activating domains of Bc-TubZ, Bt-TubZ, and Cb-TubZ reveals that no structural reorganization occurs in their C-terminal domains. However, H9 and H10 are structurally divergent among the three TubZ proteins. Because the N-terminal portion of H10 is predicted to participate in the protofilament contacts in Bt-TubZ, the subtle conformational change may affect the subunit-subunit interactions in protofilament formation (
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ).
      Figure thumbnail gr2
      FIGURE 2Structural comparison of Bc-TubZ with Bt-TubZ and Cb-TubZ (A) and with M. jannaschii FtsZ (B). A, superposition of GTPγS-Bc-TubZΔ (green), GTPγS-Bt-TubZ (cyan; Protein Data Bank code 3M89), and apo-Cb-TubZ (purple; code 3V3T). GTPγS is shown as sticks. B, superposition of GDP-Bc-TubZΔ (green) and FtsZ (pink; code 1W59). The H0 and H1 helices are shown as cylinders. Structures are superimposed using the N-terminal GTPase domain. The views are in the same orientation as in A. Mj, M. jannaschii.
      Although the subunit-subunit interactions are partly stabilized by H0 in M. jannaschii FtsZ, H0 in Bc-TubZ bridges the gap between the N- and C-terminal domains mainly by hydrophobic interactions (supplemental Fig. S5) (
      • Löwe J.
      • Amos L.A.
      Crystal structure of the bacterial cell division protein FtsZ.
      ). Bc-TubZΔ H0 is attached to H7 in a parallel manner, which is similar to Bt-TubZ H0, although the N terminus of the two Bacillus TubZ proteins shows weak sequence homology (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ,
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). Furthermore, our limited proteolytic analyses showed that the N terminus of Bc-TubZ is resistant to proteases (data not shown), indicating that H0 possesses an ordered structure that forms the extensive hydrophobic core of TubZ.

      Longitudinal Contacts between GDP-Bc-TubZΔ Molecules

      The presence of GDP in Bc-TubZΔ stabilizes the residues around the nucleotide-binding site. The GTPase domain of GDP-Bc-TubZΔ interacts with the C-terminal domain of the adjacent subunit, which makes a semicontinuous single-stranded protofilament-like arrangement in the crystals (Fig. 3A). This head-to-tail association of GDP-Bc-TubZΔ is consistent with the crystal structures of FtsZ and Bt-TubZ, in which the proteins form a longitudinal subunit repeat (
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ,
      • Oliva M.A.
      • Cordell S.C.
      • Löwe J.
      Structural insights into FtsZ protofilament formation.
      ). The contact of GDP-Bc-TubZΔ with two longitudinally neighboring molecules is 1246 Å2, covering ∼26% of the subunit surface. The C-terminal domain of chain B buries 976 Å2 of the upper surface of chain A. The interface contains a number of hydrogen bonds and hydrophobic interactions with a molecular spacing of 45 Å. This 45-Å molecular spacing is very similar to that in a previous study examining the structure of Bt-TubZ (
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). Together with the evidence that GTPγS was hydrolyzed in the crystal structure of GDP-Bc-TubZΔ, this observation suggests that the subunit-subunit interaction with the 45-Å molecular spacing reflects the protofilament contact. On the other hand, the subunit contact on the opposite side of chain A is 270 Å2 with a molecular spacing of 52 Å, which is too far for the protein-protein interactions. The arrangement of the molecules of the 52-Å contact is considered to be a crystallographic artifact because the molecular interaction with the 45-Å spacing gives a slight bend to the dimer formation, and hence, a molecular gap is required for crystallization.
      Figure thumbnail gr3
      FIGURE 3Protofilament interaction in Bc-TubZ. A, semicontinuous GDP-Bc-TubZΔ protofilament structure in the crystal lattice. The N-terminal GTPase domain is shown in green, the H7 helix in yellow, and C-terminal domain in magenta. The molecular spacing of 45 Å is shown in the middle, whereas the subunit interfaces with less interaction at the are shown at the top and bottom (52-Å spacing). Note that the 45-Å spacing is similar to that observed in the Bt-TubZ crystal structure. The regions containing the subunit-subunit interactions with the 45-Å spacing are boxed. B–D, stereo views of the GDP-binding site, the vicinity of Phe-313, and the C-terminal tail region, respectively. Residues described under “Results” are shown as sticks. In D, Arg-333 is exposed to solvent and makes few contacts with residues in H11. E and F, structural comparison of the GTPγS-Bt-TubZ and FtsZ dimers, respectively. The dimers of GTPγS-Bt-TubZ (chains D and E) (E) and FtsZ (F) observed in the crystal structures are shown. The orientation of the molecules and the color scheme are the same as for GDP-Bc-TubZΔ in A. The lower subunits are superimposed. The GTPγS-Bt-TubZ dimer is twisted, whereas GDP-Bc-TubZΔ and FtsZ are not.
      Soaking the GDP-Bc-TubZΔ crystals with GTPγS was successful only for the chain B molecule. The conformation of the loops surrounding the nucleotide is essentially the same between the GDP-Bc-TubZΔ and GTPγS-Bc-TubZΔ structures, except that the oxygen atoms of the γ-phosphate group in GTPγS make hydrogen bonds with the backbone nitrogen atoms of Gly-124 and Thr-125 in the T4 loop (supplemental Fig. S1B). This observation is consistent with the previous structures of Bt-TubZ and FtsZ showing that the protein fold itself is the same in the apo-, GDP-, and GTP-bound forms. The GDP molecule in chain A is buried in the GTP-binding pocket and covered by the T7 loop of chain B, which makes nucleotide exchange difficult. This explains why the nucleotide in Ba-TubZ polymers is GDP (
      • Chen Y.
      • Erickson H.P.
      In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.
      ). The subunit-subunit interface of the 976-Å2 contact is robust. There are three unique contacts that resemble the subunit-subunit interactions found in FtsZ and Bt-TubZ. The T7 loop, which mediates GTP hydrolysis but not nucleotide binding, covers the vicinity of GDP in the lower subunit and enables the flanking H8 helix to interact with the T3 loop by forming a salt bridge between Glu-238 and Arg-85 (Fig. 3B). In the crystal structure of GTPγS-Bt-TubZ, Arg-87 (Arg-85 in B. cereus) forms a hydrogen bond with the γ-phosphate group of GTPγS and is suggested to be a key residue in the nucleotide switch mechanism (
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). Glu-238 is one of the conserved acidic residues in the FtsZ/tubulin superfamily proteins that catalyzes GTP hydrolysis. The subunit-subunit interaction of GDP-Bc-TubZΔ in the post-GTP hydrolysis state is somewhat stabilized by the hydrogen bonds not only between Arg-85 and Glu-238 but also between Arg-85 and Glu-332 in the S9 strand. Although Arg-85 and Glu-238 are conserved in Bt-TubZ (Asp-269 in B. thuringiensis corresponding to Glu-238), Glu-332 is replaced with Gly-358 in B. thuringiensis. In the GTPγS-Bt-TubZ structure, the flanking residue Lys-359 forms a hydrogen bond with the γ-phosphate of the nucleotide. This basic residue presumably stabilizes the transition state of GTP hydrolysis, which is also seen in the RhoA-RhoGAP system (
      • Rittinger K.
      • Walker P.A.
      • Eccleston J.F.
      • Smerdon S.J.
      • Gamblin S.J.
      Structure at 1.65 Å of RhoA and its GTPase-activating protein in complex with a transition state analog.
      ). In GDP-Bc-TubZΔ, Arg-333 is positioned similarly to Lys-359 in B. thuringiensis but does not form any intra- or intermolecular contacts with any other residues. Moreover, few basic residues are capable of catalyzing the phosphoryl transfer reaction in the vicinity of the phosphate groups, which indicates that further structural analysis of the GTP-bound form is required to elucidate the GTP hydrolysis mechanism of Bc-TubZ. The H10-S9 loop is called the M loop, which is involved in subunit-subunit interactions and has the most divergent sequence among the FtsZ/tubulin family proteins. Our finding shows that the assembly mechanism is not strictly conserved between the TubZ proteins.
      The N-terminal portion of S9 makes contacts with the sugar-binding loop T5 in the lower subunit. T5 is further stabilized by H10 in the upper subunit, with a hydrogen bond between Arg-152 and Glu-320 (Fig. 3B). H10 creates a hydrophobic environment together with the H6-H7 loop in the lower subunit. Phe-313 is inserted into the hydrophobic groove formed by Tyr-189, Leu-200, and Tyr-204 and makes non-polar contacts with the aliphatic atoms of Glu-193 (Fig. 3C). Leu-200 bridges the gap between the T5 and T7 loops by contacting Phe-229, suggesting that T5 contributes to the subunit-subunit interactions in a cooperative manner with T7. The third contact exists at the C-terminal H11 helix, although the C-terminal end of Bc-TubZ was trimmed by the protease for crystallization. H11 points to the C-terminal domain of the adjacent subunit, where the aliphatic atoms of Arg-380 make hydrophobic contacts with Tyr-329 in the H10-S9 loop of the upper subunit (Fig. 3D). The H11 surrounding surface on the upper subunit is highly acidic and is possibly involved in the association with the basic C-terminal tail, which was trimmed for the crystallographic analyses (supplemental Figs. S3 and S6). The tail of Bc-TubZ likely stabilizes the protofilament as well as the monomer structure and provides the binding site for DNA that may interact with Bc-TubR encoded in the tubRZ operon (
      • Anand S.P.
      • Akhtar P.
      • Tinsley E.
      • Watkins S.C.
      • Khan S.A.
      GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.
      ).
      It should be noted that, although the monomer structures of Bc-TubZΔ and Bt-TubZ are very similar to each other, the filamentous structure of GTPγS-Bt-TubZ shows a right-handed twist, whereas the dimer structure of GDP-Bc-TubZΔ has no twist (Fig. 3, A and E). The subunit orientation of GDP-Bc-TubZΔ resembles more closely that of the FtsZ dimer (Fig. 3F). There are some possible reasons for the difference of the twists between the two TubZ proteins. One may consider that, because GDP-Bc-TubZΔ is in the post-GTP hydrolysis state, Bc-TubZ may change the subunit arrangement when it hydrolyzes GTP. Another possibility is that the Bc-TubZΔ dimer may be distorted because of crystal packing. The biochemical examination of the subunit interface is required for Bc-TubZ.

      GTPase Activation Switch

      To characterize Bc-TubZ polymerization activity, we first determined the critical concentration of Bc-TubZ needed for GTP hydrolysis (supplemental Fig. S7). Bc-TubZ at different concentrations was incubated in the polymerizing buffer, and the change in NADH concentration was measured by its absorbance at 340 nm. The critical concentration was 0.18 μm, which is very similar to that previously reported for Ba-TubZ (0.2–0.4 μm) (
      • Chen Y.
      • Erickson H.P.
      In vitro assembly studies of FtsZ/tubulin-like proteins (TubZ) from Bacillus plasmids: evidence for a capping mechanism.
      ). To test the effect of an artificial tag, we next measured the GTP hydrolysis and self-assembly levels of Bc-TubZ with a C-terminal histidine tag (Bc-TubZ-His) (Fig. 4A). Bc-TubZ-His showed a similar level of activity as the wild-type protein, consistent with the previous electron microscopy study of Bt-TubZ (
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). On the other hand, the Bc-TubZΔ construct showed little activity at 2 μm, indicating that the C terminus is critical for GTP hydrolysis. In solution, Bc-TubZΔ is unstabilized because of the lack of the C-terminal tail and is deficient in forming polymers. In the GDP-Bc-TubZΔ crystal lattice, despite the fact that the C-terminal tail is deleted, the subunit-subunit contact is tightly arranged and results in GTP hydrolysis. Our data imply that the C terminus of Bc-TubZ plays a role in fine-tuning TubZ assembly. Structural analysis of Bt-TubZ also confirms this observation, where Bt-TubZ lacking the C-terminal tail forms polymers at high concentrations (10–50 μm) but tends to aggregate readily (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ).
      Figure thumbnail gr4
      FIGURE 4Bc-TubZ GTPase activity and self-assembly. A, GTPase activity of Bc-TubZ, a Bc-TubZ fusion protein with a C-terminal histidine tag (Bc-TubZ-His), and Bc-TubZΔ. Measurements were carried out at protein concentrations of 2 μm. B, the kinetics of Bc-TubZ polymerization were monitored by measuring the increase in light scattering. A protein concentration of 2 μm was used.
      Because the polymerization activity of FtsZ is coupled to the GTP hydrolysis rate, we examined whether the polymerization is related to the GTP hydrolysis activity (
      • Mukherjee A.
      • Lutkenhaus J.
      Dynamic assembly of FtsZ regulated by GTP hydrolysis.
      ). We performed light scattering assays for Bc-TubZ and its mutants in the presence of 0.5 mm GTP and 5 μm GTPγS (Fig. 4B). The wild-type protein showed a rapid increase in turbidity up to ∼60 s, followed by a gradual decrease. Consistent with the GTPase activity measurements, the C-terminal histidine tag did not affect assembly, whereas Bc-TubZΔ completely abolished polymerization, which indicates that GTP hydrolysis of Bc-TubZ is coupled to self-assembly.
      Our GDP-Bc-TubZΔ structure suggests that the hydrogen bonds between Arg-85 and two glutamate residues (Glu-238 and Glu-332) are crucial for both the subunit-subunit interactions and GTP hydrolysis. To inspect the protofilament model of Bc-TubZ, we constructed three point mutants (R85A, E238A, and E332A) of Bc-TubZ-His and examined their GTP hydrolysis levels (Fig. 5A). The GTPase activity of the R85A mutant was completely abolished, consistent with previous reports showing that the T3 loop makes contacts with the γ-phosphate group and acts as a GTP hydrolysis switch (
      • Díaz J.F.
      • Kralicek A.
      • Mingorance J.
      • Palacios J.M.
      • Vicente M.
      • Andreu J.M.
      Activation of cell division protein FtsZ. Control of switch loop T3 conformation by the nucleotide γ-phosphate.
      ,
      • Leung A.K.
      • Lucile White E.
      • Ross L.J.
      • Reynolds R.C.
      • DeVito J.A.
      • Borhani D.W.
      Structure of Mycobacterium tuberculosis FtsZ reveals unexpected, G protein-like conformational switches.
      ). The E238A mutation in T7 abrogated the GTPase activity as well. However, this mutant assembled into polymers two times more effectively than the wild type (Fig. 5B), which indicates that E238A forms stable polymers but is deficient in GTP hydrolysis. This result is in agreement with the previous analysis of Bt-TubZ in vivo, demonstrating that Bt-TubZ(D269A), equivalent to Bc-TubZ(E238A), assembles into stable filaments in the cell but results in severe plasmid segregation defects (
      • Larsen R.A.
      • Cusumano C.
      • Fujioka A.
      • Lim-Fong G.
      • Patterson P.
      • Pogliano J.
      Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.
      ). The E332A mutant retained 40% of the original activity in both the GTPase and polymerization analyses. Indeed, the critical concentration of the E332A mutant was increased by 3-fold to 0.52 μm. On the other hand, a mutation in the flanking residue Arg-333 (R333A-His) led to similar levels compared with the wild type, indicating that Glu-332, but not Arg-333, is involved in the GTP hydrolysis reaction. Thus, our data show that the M loop participates in protofilament formation, stabilizes the filament structure, and supports GTP hydrolysis, although the residues involved in the process differ between the two species.
      Figure thumbnail gr5
      FIGURE 5GTP hydrolysis and self-assembly of Bc-TubZ mutants. A, the GTPase activities of 2 μm Bc-TubZ-His and its mutants were measured. The R85A and E238A mutations abolished the GTPase activity, whereas the activity of Bc-TubZ-His(E332A) was 40% of that of the wild type. The R333A mutation had no effect on GTPase activity. B, light scattering analyses of Bc-TubZ-His mutants. The relative activity of each reaction was determined by comparing the turbidity after a 60-s incubation. The polymerization activity was consistent with the GTPase activity, except for Bc-TubZ-His(E238A).

      Plasmid Partitioning

      Although there are still arguments as to whether TubZ contributes to replication or segregation of the Bacillus virulence plasmids, studies have shown that the polymerization activity of TubZ is essential for plasmid stability (
      • Tinsley E.
      • Khan S.A.
      A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis.
      ,
      • Tang M.
      • Bideshi D.K.
      • Park H.W.
      • Federici B.A.
      Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis.
      ,
      • Larsen R.A.
      • Cusumano C.
      • Fujioka A.
      • Lim-Fong G.
      • Patterson P.
      • Pogliano J.
      Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.
      ). Because TubZ displays a high level of GTPase activity, polymerizes into a double helical filament, and treadmills in vivo, it has been suggested that TubZ works as a cytomotive filament, such as the type II plasmid partitioning ATPase ParM, i.e. TubZ assembles into filaments to produce a driving force and achieves partitioning by pushing plasmids attached to the growing ends of filaments (
      • Aylett C.H.
      • Wang Q.
      • Michie K.A.
      • Amos L.A.
      • Löwe J.
      Filament structure of bacterial tubulin homolog TubZ.
      ). In the parMRC system, ParM forms a stable polymer in the presence of the ParR-parC complex in vivo (
      • Garner E.C.
      • Campbell C.S.
      • Mullins R.D.
      Dynamic instability in a DNA-segregating prokaryotic actin homolog.
      ). By binding to parC, ParR plays a dual role in transcriptional repression and segrosome organization of the R1 plasmid, which provides a nucleation site for the assembly of ParM (
      • Jensen R.B.
      • Dam M.
      • Gerdes K.
      Partitioning of plasmid R1. The parA operon is autoregulated by ParR, and its transcription is highly stimulated by a downstream activating element.
      ,
      • Garner E.C.
      • Campbell C.S.
      • Weibel D.B.
      • Mullins R.D.
      Reconstitution of DNA segregation driven by assembly of a prokaryotic actin homolog.
      ). In contrast, TubZ polymerization is not dependent on TubR but rather on TubZ protein concentration in vivo (
      • Larsen R.A.
      • Cusumano C.
      • Fujioka A.
      • Lim-Fong G.
      • Patterson P.
      • Pogliano J.
      Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis.
      ,
      • Akhtar P.
      • Anand S.P.
      • Watkins S.C.
      • Khan S.A.
      The tubulin-like RepX protein encoded by the pXO1 plasmid forms polymers in vivo in Bacillus anthracis.
      ). Although the function of TubR in the type III plasmid segregation system is not yet clear, given that Bt-TubR represses the transcription of the tubRZ operon and forms a ternary complex with the centromeric DNA and Bt-TubZ, one may speculate that Bt-TubR forms a pBtoxis segrosome and recruits Bt-TubZ to segregate the plasmid into the daughter cells (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ). To test the interaction between Bc-TubR and Bc-TubZ polymers, we carried out sedimentation of polymerized Bc-TubZ with or without Bc-TubR (Fig. 6A). Bc-TubR was co-sedimented with Bc-TubZ polymers in vitro but did not enhance Bc-TubZ sedimentation. This result was confirmed by the GTPase activity analyses of Bc-TubZ with Bc-TubR, which showed that Bc-TubR did not promote the GTPase activity of Bc-TubZ (Fig. 6B). Consistent with this, negative-stain EM images showed that, in the presence of Bc-TubR, Bc-TubZ formed similar polymers compared with Bc-TubZ alone (supplemental Fig. S8). Co-sedimentation of Bc-TubR with Bc-TubZ-His polymers was also examined because the C-terminal tail of Bt-TubZ is critical for ternary complex formation between Bt-TubZ, Bt-TubR, and DNA. The additional tag at the C terminus of Bc-TubZ abolished the interaction with Bc-TubR. These results indicate that Bc-TubR interacts with Bc-TubZ polymers and that the Bc-TubZ tail is required for Bc-TubR association. We further examined the GTPase activity of Bc-TubZ in the presence of Bc-TubR and the plasmid carrying the tubRZ operon. Unlike the parMRC system, the complex of Bc-TubR and the plasmid did not stimulate the GTPase activity of Bc-TubZ, which suggests either that the GTPase activity is not essential or that other cellular components are required for the activation of the cytoskeletal function of Bc-TubZ.
      Figure thumbnail gr6
      FIGURE 6Effect of Bc-TubR and DNA in Bc-TubZ polymerization. A, Bc-TubR binding to Bc-TubZ polymers. Sedimentations of Bc-TubZ and Bc-TubZ-His were performed in the presence or absence of Bc-TubR. BSA binding was also examined as a negative control. B, GTPase activity of 2 μm Bc-TubZ in the presence or absence of 2 μm Bc-TubR and 3 nm pBS_Bc-tubRZ.

      DISCUSSION

      Although our data show that the residues involved in the GTP hydrolysis mechanism are not strictly conserved between Bc-TubZ and Bt-TubZ, their overall structural properties resemble each other. A significant feature of TubZ lies in the C-terminal tail, which plays a critical role in both the structural stability of TubZ and TubR association. The previous electron microscopy study of the Bt-TubZ filament showed that the C-terminal tail is exposed to solvent, which is consistent with our proteolytic analysis of the Bc-TubZ polymer, demonstrating that the C-terminal tail is sensitive to thermolysin (data not shown). The tail of TubZ comprises several basic residues and likely binds both TubR and DNA. Moreover, our Bc-TubZ sedimentation analysis suggests that Bc-TubR is capable of associating with the Bc-TubZ filament in the absence of DNA, raising the question of whether Bc-TubR recognizes the specific site of Bc-TubZ filaments. One possibility is that centromeric DNA may define the higher order structure of the segrosome by Bc-TubR association, and thus, the segrosome can recognize distinct sites of the Bc-TubZ filament at a specific location in the cell. With regard to the TubR recognition sequence, although the binding sequence of Bt-TubR has been identified as four 12-bp imperfect direct repeats located upstream of the tubR gene, no similar sequence has been found in the corresponding region in pXO1 (
      • Tang M.
      • Bideshi D.K.
      • Park H.W.
      • Federici B.A.
      Iteron-binding ORF157 and FtsZ-like ORF156 proteins encoded by pBtoxis play a role in its replication in Bacillus thuringiensis subsp. israelensis.
      ). Instead, a 24-bp inverted repeat downstream of the tubZ gene has been predicted to be the putative centromeric DNA in B. anthracis (
      • Tinsley E.
      • Khan S.A.
      A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis.
      ). Because there are certain varieties of the centromere-binding proteins in the plasmid partition systems, the molecular mechanisms may not be simple, even among virulent Bacillus species. Finally, the segregation model has been proposed for plasmid transport by TubZ; the TubZ filament works as a tram and gives the plasmid a ride to its destination by interacting with TubR (
      • Ni L.
      • Xu W.
      • Kumaraswami M.
      • Schumacher M.A.
      Plasmid protein TubR uses a distinct mode of HTH-DNA binding and recruits the prokaryotic tubulin homolog TubZ to effect DNA partition.
      ). If this is the case, how does the complex composed of TubR and the plasmid recognize the destination and detach from the tram? Mechanistic details of the interaction between the segrosome and the TubZ filaments will provide insights into the new plasmid partition system and the novel function of the cytoskeletal proteins.

      Acknowledgments

      We thank the NW12A beamline staff of Photon Factory Advanced Ring (PF-AR, Tsukuba, Japan) and the BL26B2 beamline staff of SPring-8 (Harima, Japan) for help with data collection. We also thank Dr. Yoshifumi Nishimura for access to the circular dichroism equipment and Drs. Kiminori Toyooka and Mayuko Sato (RIKEN Plant Science Center, Kanagawa, Japan) for the EM facility.

      Supplementary Material

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