C. botulinum phage c-st partition operon and TubZ structure. (A) CST188–190 cluster structure and localization in phage c-st genome. (B) Cartoon representation of the apo TubZ-T100A structure. Secondary structure elements are labeled according to Nogales et al. (2). The nucleotide-binding domain (N-terminal domain) with an empty nucleotide-binding site is shown in blue, the GTPase domain (C-terminal domain) is shown in red, the central helix H7 is shown in yellow, and the long C-termini TubZ helix H11 is shown in orange. (C) Superimposition of phage TubZ (salmon) onto B. thuringiensis plasmid TubZ (gray, displaying bound nucleotide). Regions with extra secondary structure elements in plasmid TubZ are highlighted in purple, and those with extra secondary structure elements in loops are highlighted in blue. (D) 3D alignment of protein structures from phage (PTubZ) and bacteria (TubZ) with reference to secondary structure elements (E, strand; H, helix; L, loop) and highlighting conserved residues (gray). Colors are the same as in B.

 TubR-tubS interaction. (A) EMSAs using purified TubR and γ-³²P–labeled DNA subfragment S1-114 (Left) and B. thuringiensis centromere-like sequence tubS (Right). The DNA band shift (B, bound; UB, unbound) attributable to binding of TubR is indicated. The TubR concentrations increase fourfold in successive lanes from left to right. (B) AUC velocity sedimentation experiments, where each curve correspond to an independent experiment of 10 μM TubR (black; monitored at 280 nm), 1.2 μM subfragment S1-114 (tubS; red), or 1.2 μM tubS plus 10 μM TubR (blue), with the latter two monitored at 260 nm. The sedimentation peaks from protein (1.2S) and DNA (3.2S) are clearly distinguishable from the complex (4.6S). (C) Titration of tubS with increasing TubR concentrations. Binding was determined by the increment molecular mass of the complexes measured by sedimentation equilibrium AUC. The binding of protein to DNA increases with protein concentration to near-saturation at a stoichiometry of 3.55 ± 0.21 TubR per tubS DNA molecule, according to a cooperative model fit indicated by the line.

 Gene organization and evolution of TubZ-based segrosomes. (A) Comparative schematic diagram of the type III segregation system cluster in different replicons from Clostridium and two Bacillus species. The positions of the genes tubZ (orange), tubR (red), and the unique component described here tubY (yellow) are shown. (B) Phylogenetic tree of TubZ shows relationships between phage (red), plasmid (blue), and chromosomal (gray) tubZ sequences. Bootstrap values representing confidence levels are indicated for selected branches. (Right) Presence or absence of tubR (R) and tubY (Y) near tubZ is shown. Question marks indicate the presence of putative proteins that share with TubR only an HTH-motif and a small size or include only long coiled-coil motifs like TubY.

 Assembly properties of phage TubZ. (A) Electron micrograph of 10 μM TubZ two-stranded helical filaments assembled in PKE buffer [50 mM piperazine-1,4-bis-2-ethanesulfonic acid–potassium hydroxide, 100 mM potassium acetate, 1 mM EDTA (pH 6.5)] with 1 mM GTP and 6 mM Mg²⁺. (Scale bar: 100 nm.) (B) TubZ assembly monitored by 90° light scattering. WT TubZ (10 μM) polymerized in PKE buffer with 6 mM Mg²⁺ and 1 mM GTP (black), 0.1 mM GMPCPP (dotted black line), or 0.1 mM GTP-γ-S (gray). TubZ-T100A (10 μM; orange) or TubZ-E200A (10 μM; blue) was assembled in PKE with 6 mM Mg²⁺ and 1 mM GTP. There is an increase/decrease on the signal attributable to the formation/disassembly of polymers. (C) SDS/PAGE of sedimentation assays of 10 μM TubZ (WT and mutants) assembled in PKE buffer with 6 mM Mg²⁺ and 1 mM GTP (line 1), 2 mM GDP (line 2), 0.1 mM GMPCPP (line 3), 0.1 mM GMPCP (line 4), and 0.1mM GTP-γ-S (line 5). P, pellet; S, supernatant. Nucleotide diphosphates GDP and GMPCP do not induce filament formation.

 TubR and TubY interaction with TubZ filaments. (A) TubZ (10 μM) polymerization monitored by 90° light scattering (black) shows the effect of the partners’ addition during assembly. Additions are 10 μM TubR-5 μM tubS complex (red), 10 μM TubY₂₂₆ (yellow), 10 μM TubY₁₃₇ (blue), and 5 μM tubS (dotted green line). (B) Electron micrograph of TubZ bundles induced by interaction with TubR-S. (C) Titration by sedimentation of the increase in TubZ polymerization vs. TubR–tubS complex concentration. The solid line is a model fit for approximately 1:1 binding with K_d = 39 μM. (D) TubZ filaments in the presence of TubY_226. (E) Titrations by sedimentation of the increase in TubZ polymerization vs. TubY₂₂₆ concentration; the line is a model fit with 1:1 binding (n = 1) and K_d = 0.9 μM. Models with n < 1 were equally compatible with the data, whereas models with n > 1 strongly deviated from the data. (F) Reshaping of the TubZ-R-S complex bundles into rings when TubY₂₂₆ is added. (Scale bars: 100 nm.)